Aminoglycoside antibiotics targeting bacterial 16s ribosomal rna

ABSTRACT

The present invention relates to paromamine-based compounds according to formula I having selective antimicrobial activity directed at ribosomal 16S RNA. Furthermore, the invention is directed to the use of said compounds for preparing a medicament, pharmaceutical preparations and methods for preparing said compounds

FIELD OF THE INVENTION

The present invention relates to paromamine-based compounds according toformula I having selective antimicrobial activity directed at ribosomal16S RNA. Furthermore, the invention is directed to the use of saidcompounds for preparing a medicament, pharmaceutical preparations, andmethods for preparing said compounds.

BACKGROUND OF THE INVENTION

Aminoglycoside antibiotics (AGAs) are clinically important drugseffective against a broad range of microorganisms. The clinical use ofAGAs is restricted by toxicity (irreversible ototoxicity and reversiblenephrotoxicity) and by the resistance of pathogens to AGAs. Common to2-deoxystreptamine derived AGAs is a pseudodisaccharide core of theneamine type. It is composed of 2-deoxystreptamine (ring II)glycosidically linked to an aminodeoxyglucopyranose (ring I). Additionalglycosyl moieties are attached to the hydroxy groups of the2-deoxystreptamine moiety to give rise to a variety of compounds,categorized as 4,5- or 4,6-substituted deoxystreptamine-derivedaminoglycosides, such as paromomycin (1a) and kanamycin A (1b).

AGAs affect the fidelity of protein synthesis through binding tospecific sites of the ribosomal RNA (rRNA) (Magnet et al., Chem. Rev.2005, 105, 477; Jana et al., Appl. Microbiol. Biotechnol. 2006, 70, 140;Vicens et al., Chembiochem 2003, 4, 1018; Ogle et al., Trends BiochemSci 2003, 28, 259). In spite of decades of use of ribosomal drugs, thestructural features governing selectivity, i.e. the discriminationbetween prokaryotic and eukaryotic ribosomes, and the toxicity of thesecompounds are still not fully understood. Genetic studies (Hobble etal., Antimicrob. Agents Chemother. 2006, 50, 1489; Hobbie et al., C.Antimicrob. Agents Chemother. 2005, 49, 5112; Boettger et al., EMBOreports 2001, 2, 318) and crystal structures of AGAs complexed withribosomal subunits (Carter et al. Nature 2000, 407, 340; Francois et al.Nucleic Acids Res 2005, 33, 5677; and above mentioned references) havecontributed to understanding the interactions of AGAs with the rRNAtarget.

The above-mentioned studies have shown that the aminodeoxyglucopyranosylring I of 2-deoxystreptamine-derived AGAs binds to the rRNA in the sameway regardless of whether the 2-deoxystreptamine is 4,5- or4,6-disubstituted. According to the crystal structures of several AGAs,ring I intercalates into the bulge formed by A1408, A1492 and A1493, andthe base pair C1409-G1491. Ring I stacks upon G1491 and forms a pseudobase pair with A1408 characterized by H-bonds from C(6′)-OH to N(1) ofA1408, and from C(5′)-OH to N(6) of A1408. Additionally, ring I showstwo non-specific interactions with the phosphate groups of the twoflipped-out adenines 1492 and 1493: C(3′)-OH forms an hydrogen bond withO2P of A1492, and C(4′)-OH forms a hydrogen bond with O2P of A1493.

Neamine-based derivatives are currently under investigation for reducingbacterial) aminoglyoside resistance and for use as anti-HIV agents.

U.S. patent application 2006/0211634 A1 teaches the use of neamine-basedcompounds for inhibiting aminoglycoside-6″-N-acetyltransferases capableof reversing or inhibiting bacterial resistance to aminoglycosideantibiotics. These compounds are characterized by large substituents onthe 6″position such as Coenzyme A. They are not suggested for use asantibiotics.

WO 2005/060573 teaches compositions for modulating the activity of anucleic acid molecule comprising a peptide nucleic acid moietyconjugated to a neamine moiety. The document does not discloseantibiotic activity for these compositions.

Feng et al. (Angew. Chem. Int. Ed. 2005, 44, 6859-6862) discloses theregio- and chemoselective 6′-N-derivatisation of neamine-basedaminoglycosides with coenzyme A resulting in bisubstrate inhibitors asprobes for studying aminoglycoside 6′-N-acetyltransferases (AAC(6′)inhibitors). The same authors (Feng et al., J. Med. Chem. 2006, 4,5273-5281) describe second generation AAC(6′) inhibitors based onneamine having long polypeptidic substituents in the 6′ position.

Riguet et al. (Tetrahedron 60, 2004: 8053-8064) teach a route forpreparing neamine-based derivatives with heterocyclic substituents boundby linker units for targeting HIV1 TAR RNA. Later the same authors teach(Bioorganic & Medicinal Chemistry Letters 15 (2005) 4651-4655)neamine-based dimers and trimers for targeting HIV-1 TAR RNA.

Due to their high toxicity and significant levels of antibioticresistance neamine-based aminoglycosides are presently of limited use.

The object underlying the present invention is to provide novel andimproved antimicrobial compounds that are not modified by commonmicrobial resistance determinants and that target microbial, inparticular bacterial 16S ribosomal RNA, i.e. the compounds do not targetat all or target to a substantially less degree eukaryotic cytosolicand/or mitochiondrial ribosomes.

DESCRIPTION OF THE INVENTION

It was found that specific paromamine-based compounds selectively targetmicrobial 16 S RNA.

In a first aspect the present invention relates to compounds of formula(I):

wherein:X, Y and Z denote in each case, independently of one another, —O—, —NH—,—S—, substituted or unsubstituted —CH₂— or a direct bond to R¹ and/orR²;R¹ and R² denote in each case, independently of one another, hydrogen,linear or branched, substituted or non-substituted alkyl, alkenyl,alkynyl, alkylidene, carbocycle, or YR¹ and ZR² together form asubstituted or non-substituted cycloalkyl or a correspondingheterocyclic ring;R³ and R⁴ denote in each case, independently of one another, hydrogen,amino or hydroxyl;R⁵ and R⁶ denote in each case, independently of one another, hydrogen orglycosyl) residues;and their diastereoisomers or enantiomers in the form of their bases orsalts of physiologically acceptable acids.

In the context of the present invention it is understood that antecedentterms such as linear or branched, substituted or non-substitutedindicate that each one of the subsequent terms is to be interpreted asbeing modified by said antecedent term. For example, the scope of theterm “linear or branched, substituted or non-substituted alkyl, alkenyl,alkynyl, alkylidene, carbocycle” encompasses linear or branched,substituted or non-substituted alkyl; linear or branched, substituted ornon-substituted alkenyl; linear or branched, substituted ornon-substituted alkynyl; linear or branched, substituted ornon-substituted alkylidene; and linear or branched, substituted ornon-substituted carbocycle. For example, the term “C₂-C₁₂ alkenyl,alkynyl, or alkylidene” indicates the group of compounds having 2 to 12carbons and alkenyl, alkynyl, or alkylidene functionality.

The compounds of the present invention are stable and resistant tobacterial degradation. However, it was demonstrated that some preferredembodiments of the compounds are more resistant against bacterialdeterminants than others.

In a preferred embodiment of the present invention the compounds offormula I are those, wherein R¹ and/or R², preferably R¹ and R² are nothydrogen. Said compounds demonstrate either a partial (R¹ and/or R²≠H)or a complete (R¹ and R² ≠H) resistance against resistance determinantsselected from the group consisting of ANT4′-OH, APH2″-OH/AAC6′-NH,AAC3-NH, ANT2″-OH, AAC6′-NH (Magnet et al., Chem. Rev. 2005).

In a more preferred embodiment the compounds of formula I are those,wherein YR¹ and ZR² together form a substituted or non-substitutedcycloalkyl or a corresponding heterocyclic ring. Most preferred saidcycloalkyl is an alkylidene, preferably an arylalkylidene (such as abenzylidene), most preferably an arylalkylidene substituted in the arylring.

In another preferred embodiment of the present invention the compoundsof formula I are those, wherein R³ and/or R⁴, preferably R³ and R⁴ arehydrogen, i.e. they are not amino or hydroxyl. Said compoundsdemonstrate a resistance against one or both resistance determinantsAPH3′ (if R³ is H) and AAC2′ (if R⁴ is H).

In a most preferred embodiment, the compounds of formula I are those,wherein R¹ and/or R² are not hydrogen and R³ and/or R⁴ are hydrogen.

In a preferred embodiment of the present invention R¹ and R² denote ineach case, independently of one another, hydrogen, linear or branched,substituted or non-substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, alkynyl oralkylidene, C₃-C₁₂ cycloalkyl, C₃-C₂₀ aryl, preferably arylalkyl, C₃-C₂₀heteroaryl or C₃-C₂₀ heterocyclic residues. More preferably, R¹ and R²denote in each case, independently of one another, linear or branched,substituted or non-substituted C₁-C₈ alkyl, C₂-C₈ alkenyl, alkynyl oralkylidene, C₅-C₁₂ cycloalkyl, C₅-C₁₂ cycloaryl, C₅-C₁₂ heteroaryl orC₅-C₁₂ heterocyclic residues. Most preferably, R¹ and R² denote in eachcase, independently of one another, linear or branched, substituted ornon-substituted C₁-C₄ alkyl, C₂-C₆ alkenyl, alkynyl or alkylidene, C₅-C₆cycloalkyl, C₅-C₆ cycloaryl, C₅-C₆ heteroaryl or C₅-C₆ heterocyclicresidues.

In a preferred embodiment YR¹ is not NH₂. In another preferredembodiment ZR² is not OH.

It is also preferred that R¹ and/or R² do not comprise peptide nucleicacid moieties.

It is noted that increased size and steric hindrance in R¹ and/or R² canreduce selectivity and/or increase toxicity. Generally speaking, smallerR¹ and/or R² substituents are preferred for that reason.

Preferably, R¹ comprises, preferably is, an alkylaryl group, substitutedor non-substituted in the aryl moiety, preferably a substituted ornon-substituted (C₁-C₅ alkyl)aryl group and R² is H.

In another preferred embodiment R² is a linear or branched, substitutedor non-substituted C₁-C₇ alkyl, C₂-C₇ alkenyl, alkynyl, or alkylidene,C₃-C₇ cycloalkyl, C₃-C₇ aryl, preferably aralkyl, C₃-C₇ heteroaryl orC₃-C₇ heterocyclic and R¹ is hydrogen.

In another preferred embodiment of the present invention R³ is hydroxyl.

In another preferred embodiment of the present invention R⁴ is amino.

In another preferred embodiment of the present invention R⁵ is hydrogen.

In a preferred embodiment of the present invention R⁵ is selected fromthe group consisting of mono- and polysaccharides. Preferably R⁵ is amono-, di- or trisaccharide, more preferably a mono- or disaccharide,most preferably a disaccharide, especially preferred a2,6-diamino-2,6-dideoxy-β-L-idopyranosyl-(1→3)-β-D-ribofuranosyl moiety.

Preferably, R⁶ is hydrogen.

In another preferred embodiment R⁶ is selected from the group consistingof mono- and polysaccharides, preferably a mono-, di- or trisaccharide,more preferably a mono- or disaccharide, most preferably amonosaccharide, especially preferred a3-amino-3-deoxy-α-D-glucopyranosyl moiety.

In another preferred embodiment R⁶ is a2,6-diamino-2,6-dideoxy-β-L-idopyranosyl-(1→3)-β-D-ribofuranosyl moiety.

In another more preferred embodiment YR¹ and ZR² together form asubstituted or non-substituted cycloalkyl or a correspondingheterocyclic ring. Preferably, the ring formed by YR¹ and ZR² and ring Iof formula I is a five or six-membered ring, more preferably asix-membered ring.

Most preferably, YR¹ and ZR² together form a 6-membered 4′,6′-cycloalkylor substituted 4′,6′-cycloalkyl ring.

In another preferred embodiment Y and Z, preferably X, Y and Z areoxygen.

In a more preferred embodiment compounds of formula (I) above arecompounds of formula (II) below:

herein:X, Y and Z denote in each case, independently of one another, —O—, —NH—,—S—, substituted or non-substituted —CH₂—;R⁷ denotes hydrogen, linear or branched, substituted or non-substitutedalkyl, alkenyl, alkynyl, alkylidene, or carbocycle;R⁸ denotes hydrogen, OH with the proviso that the compound is stable,NH₂, NR_(a)R_(b), SH, SR_(a), OR_(a) or a linear or branched,substituted or non-substituted C₁-C₈ alkyl, preferably C₁-C₄ alkyl,wherein R_(a) and R_(b) are in each case, independently of one another,C₁-C₈ alkyl, preferably C₁-C₄ alkyl;R³ denotes hydrogen, amino or hydroxyl, preferably amino or hydroxyl;R⁴ denotes hydrogen, amino or hydroxyl, preferably amino or hydroxyl;R⁵ denotes hydrogen, a mono- or polysaccharide, preferably a mono-, di-or trisaccharide, more preferably a mono- or disaccharide, mostpreferably a disaccharide, especially preferred a2,6-diamino-2,6-dideoxy-β-L-idopyranosyl-(1→3)-β-D-ribofuranosyl moiety;R⁶ denotes hydrogen, a mono- or polysaccharide, preferably a mono-, di-or trisaccharide, more preferably a mono- or disaccharide, mostpreferably a monosaccharide, especially preferred a3-amino-3-deoxy-α-D-glucopyranosyl moiety.

In formula II X, Y and Z are preferably all oxygen.

In a preferred embodiment of formula II R⁷ denotes hydrogen, a linear orbranched, substituted or non-substituted C₁-C₈ alkyl, C₃-C₈ cycloalkyl,C₅-C₂₀ aryl, C₅-C₂₀ heteroaryl, preferably C₅-C₁₂ heteroaryl.

In a more preferred embodiment of formula II R⁷ denotes linear,substituted or non-substituted C₁-C₈ alkyl, preferably substitutedlinear C₁-C₃ alkyl, more preferably aryl-substituted C₁-C₃ alkyl;substituted or non-substituted C₃-C₈ cycloalkyl, C₅-C₁₂ aryl, C₅-C₁₂heteroaryl, preferably substituted C₅-C₁₂ heteroaryl.

In another more preferred embodiment R⁷ denotes C₅-C₁₂ aryl orheteroaryl, preferably a (C₁-C₇ alkyl)aryl group.

In a more preferred embodiment of formula II R⁷ is an aryl substitutedethyl group.

In another preferred embodiment compounds of the present invention offormula I or II comprise one or more halogens, preferably one halogen,preferably a chlorine, bromine, fluorine or iodine, more preferably afluorine. For compounds of formula II it is preferred that R⁷ comprisesa halogen, preferably a chlorine, bromine, fluorine or iodine, morepreferably a fluorine.

For compounds of formula II it is preferred that R⁸ is selected from thegroup consisting of hydrogen, halogen or linear or branched, substitutedor non-substituted C₁-C₈ alkyl, preferably C₁-C₄ alkyl, most preferablyhydrogen.

For compounds of formula II R⁵ or R⁶ is preferably hydrogen.

For compounds of formula II R⁶ is preferably a2,6-diamino-2,6-dideoxy-β-L-idopyranosyl-(1→3)-β-D-ribofuranosyl moiety.

In a further preferred embodiment of the invention relating to compoundsof formula I and/or II R¹ and/or R², preferably both, denote a linear orbranched, substituted or non-substituted alkyl or cycloalkyl, whereinone or more of the carbon atoms are replaced, independently of oneanother, by oxygen, sulfur or nitrogen atoms.

In a most preferred embodiment the paromamine-based compounds of thepresent invention are selected from the group consisting of

-   4′,6′-O-benzylideneparomomycin,-   4′,6′-O-p-methoxybenzylideneparomomycin,-   4′,6′-O-m-methoxybenzylideneparomomycin tetraacetate,-   4′,6′-O-o-methoxybenzylideneparomomycin,-   4′,6′-O-2,5-dimethoxybenzylideneparomomycin,-   4′,6′-O-p-nitrobenzylideneparomomycin,-   4′,6′-O-m-nitrobenzylideneparomomycin triacetate,-   4′,6′-O-p-chlorobenzylideneparomomycin,-   4′,6′-O-3,5-dichlorobenzylideneparomomycin,-   4′,6′-O-p-cyanobenzylideneparomomycin,-   4′,6′-O-p-phenylbenzylideneparomomycin,-   4′,6′-O-p-fluorobenzylideneparomomycin,-   4′,6′-O-3,5-dimethoxybenzylideneparomomycin,-   4′,6′-O-3,4,5-trimethoxybenzylideneparomomycin,-   4′,6′-O-m-chlorobenzylideneparomomycin,-   4′,6′-O-o-nitrobenzylideneparomomycin,-   4′,6′-O-p-trifluoromethylbenzylideneparomomycin,-   4′,6′-O-p-dimethylaminobenzylideneparomomycin,-   4′,6′-O-1-naphthylideneparomomycin,-   4′,6′-O-2-naphthylideneparomomycin,-   4′,6′-O-2-furanylideneparomomycin,-   4′,6′-O-2-thiophenylideneparomomycin and-   4′,6′-O-ethylideneparomomycin,-   4′,6′-O-(2-phenyl)-ethylideneparomomycin,-   4′,6′-O-(3-phenyl)-propylideneparomomycin,-   4′,6′-O-(3-phenyl)-propenylideneparomomycin,-   4′,6′-O-cyclohexylmethylideneparomomycin,-   4′-O-benzylparomomycin,-   6′-O-benzylparomomycin,-   4′-p-chlorobenzyl paromomycin,-   4′-p-(trifluoromethyl)benzylparomomycin,-   4′-benzyloxymethylparomomycin and-   4′-p-methoxybenzylparomomycin.

DEFINITIONS

In all compounds disclosed herein, in the event that the nomenclatureconflicts with the structure, it shall be understood that the compoundis defined by the structure.

The invention includes all compounds described herein containing one ormore asymmetric carbon atoms that may occur as racemates and racemicmixtures, single enantiomers, diastereoisomeric mixtures and individualdiastereoisomers. All such isomeric forms of these compounds areexpressly included in the present invention. Each stereogenic carbon maybe in the R or S configuration or a combination of configurations. It isunderstood that the stereogenic structure of the paromamine core of thecompounds of the invention is fixed as shown in formulas I, I and II.

Some of the compounds of the general formulas (I) and (II) disclosedherein can exist in more than one tautomeric form. The present inventionincludes all such tautomers.

All terms as used herein shall be understood by their ordinary meaningas known in the art.

The term “heteroatom” as used herein shall be understood to mean atomsother than carbon and hydrogen such as and preferably O, N, S and P.

The terms alkyl, alkenyl, alkynyl, alkylidene, etc. shall be understoodas encompassing linear as well as branched forms of carbon-containingchains where structurally possible. In these carbon chains one or morecarbon atoms can be optionally replaced by heteroatoms, preferably by O,S or N. If N is not substituted it is NH. The heteroatoms may replaceeither terminal or internal carbon atoms within a linear or branchedcarbon chain. Such groups can be substituted as herein described bygroups such as oxo to result in definitions such as but not limited toalkoxycarbonyl, acryl, amido and thioxo.

The term “carbocycle” shall be understood to mean an aliphatichydrocarbon radical containing from 3 to 20, preferably from 3 to 12carbon atoms, more preferably 5 or 6 carbon atoms. Carbocylces includehydrocarbon rings containing from 3 to 10 carbon atoms. Thesecarbocycles may be either aromatic or non-aromatic systems. Thenon-aromatic ring systems may be mono or polyunsaturated. Preferredcarbocycles include but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptanyl,cycloheptenyl, phenyl, indanyl, indenyl, benzocyclobutanyl,dihydronaphthyl, tetrahydronaphthyl, naphthyl, decahydronaphthyl,benzocycloheptanyl, and benzocycloheptenyl. Certain terms for cycloalkylsuch as cyclobutanyl and cyclobutyl shall be used interchangeably.

The term “cycloalkyl” shall be understood to mean aliphatichydrocarbon-containing rings having from 3 to 12 carbon atoms. Thesenon-aromatic ring systems may be mono- or polyunsaturated, i.e. the termencompasses cycloalkenyl and cycloalkynyl. The cycloalkyl may compriseheteroatoms, preferably O, S or N, and be substituted ornon-substituted. Preferred and non-limiting cycloalkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,cyclohexenyl, cycloheptanyl, cycloheptenyl, benzocyclobutanyl,benzocycloheptanyl and benzocycloheptenyl.

The term “heterocyclic” refers to a stable non-aromatic, preferably 3 to20 membered, more preferably 3-12 membered, most preferably 5 or 6membered, monocyclic or multicyclic, preferably 8-12 membered bicyclic,heteroatom-containing cyclic radical, that may be either saturated orunsaturated. Each heterocycle consists of carbon atoms and one or more,preferably 1 to 4 heteroatoms chosen from nitrogen, oxygen and sulphur.The heterocyclic residue may be bound to the remaining structure of thecomplete molecule by any atom of the cycle, which results in a stablestructure. Exemplary heterocycles include but are not limited topyrrolidinyl, pyrrolinyl, morpholinyl, thiomorpholinyl, thiomorpholinylsulfoxide, thiomorpholinyl sulfone, dioxalanyl, piperidinyl,piperazinyl, tetrahydrofuranyl, 1-oxo-λ4-thiomorpholinyl,13-oxa-11-aza-tricyclo[7.3.1.0-2,7]tridecy-2,4,6-triene,tetrahydropyranyl, 2-oxo-2H-pyranyl, tetrahydrofuranyl, 1,3-dioxolanone,1,3-dioxanone, 1,4-dioxanyl, 8-oxa-3-aza-bicyclo[3.2.1]octanyl,2-oxa-5-aza-bicyclo[2.2.1]heptanyl, 2-thia-5-aza-bicyclo[2.2.1]heptanyl,piperidinonyl, tetrahydro-pyrimidonyl, pentamethylene sulphide,pentamethylene sulfoxide, pentamethylene sulfone, tetramethylenesulphide, tetramethylene sulfoxide and tetramethylene sulfone.

The term “aryl” as used herein shall be understood to mean an aromaticcarbocycle or heteroaryl as defined herein. Each aryl or heteroarylunless otherwise specified includes its partially or fully hydrogenatedderivative. For example, quinolinyl may include decahydroquinolinyl andtetrahydroquinolinyl; naphthyl may include its hydrogenated derivativessuch as tetrahydronaphthyl. Other partially or fully hydrogenatedderivatives of the aryl and heteroaryl compounds described herein willbe apparent to one of ordinary skill in the art. Naturally, the termencompasses aralkyl and alkylaryl, both of which are preferredembodiments for practicing the compounds of the present invention. Forexample, the term aryl encompasses phenyl, indanyl, indenyl,dihydronaphthyl, tetrahydronaphthyl, naphthyl and decahydronaphthyl.

The term “heteroaryl” shall be understood to mean an aromatic C₃-C₂₀,preferably 5-8 membered monoxyclic or preferably 8-12 membered bicyclicring containing 1-4 heteroatoms such as N, O and S. Exemplaryheteroaryls comprise aziridinyl, thienyl, furanyl, isoxazolyl, oxazolyl,thiazolyl, thiadiazolyl, tetrazolyl, pyrazolyl, pyrrolyl, imidazolyl,pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyranyl, quinoxalinyl,indolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzothienyl,quinolinyl, quinazolinyl, naphthyridinyl, indazolyl, triazolyl,pyrazolo[3,4-b]pyrimidinyl, purinyl, pyrrolo[2,3-b]pyridinyl,pyrazole[3,4-b]pyridinyl, tubercidinyl, oxazo[4,5-b]pyridinyl andimidazo[4,5-b]pyridinyl.

Terms which are analogues of the above cyclic moieties such as aryloxyor heteroaryl amine shall be understood to mean an aryl, heteroaryl,heterocycle as defined above attached to its respective group.

As used herein, the terms “nitrogen” and “sulphur” include any oxidizedform of nitrogen and sulphur and the quaternized form of any basicnitrogen as long as the resulting compound is chemically stable. Forexample, for an —S—C₁₋₆ alkyl radical shall be understood to include—S(O)—C₁₋₆alkyl and —S(O)₂—C₁₋₆ alkyl.

The compounds of the invention are only those which are contemplated tobe ‘chemically stable’ as will be appreciated by those skilled in theart. For example, compounds having a ‘dangling valency’ or a ‘carbanion’are not compounds contemplated by the inventive disclosed herein.

The above described compounds have demonstrated a strong andantimicrobial, in particular antibacterial, 16 S RNA specific activitymaking them particularly useful for preparing medicaments lackingtoxicity due to the essential lack of activity in eukaryotic cells, i.e.no interaction with eukaryotic cytosolic and/or mitochondrial RNA.

Because of the above described highly selective activity another aspectof the present invention relates to the use of one or more compounds ofthe invention for preparing a medicament.

In a preferred embodiment one or more compounds of the present inventionare used for preparing a medicament for the treatment and/or preventionof a microbial, preferably a bacterial, infection.

In a further preferred embodiment the invention relates to the use ofone or more compounds according to the invention for preparing amedicament for the treatment and/or prevention of leishmaniasis.

In another preferred embodiment the invention relates to the use of oneor more compounds according to the invention for preparing a medicamentfor the treatment and/or prevention of trypanosomiasis.

A further aspect of the present invention concerns pharmaceuticalcompositions, comprising as active substance one or more compounds ofthe present invention or pharmaceuticclly acceptable derivatives orprodrugs thereof, optionally combined with conventional excipientsand/or carriers.

Medical Use and Pharmaceutical Compositions

The invention includes pharmaceutically acceptable derivatives ofcompounds of formulae (I) and (II). A “pharmaceutically acceptablederivative” refers to any pharmaceutically acceptable salt or ester orany other compound which, upon administration to a patient, is capableof providing (directly or indirectly) a compound of the invention, or apharmacologically active metabolite or pharmacologically active residuethereof. A pharmacologically active metabolite shall be understood tomean any compound of the invention capable of being metabolizedenzymatically or chemically. This includes, for example, hydroxylated oroxidized derivative compounds of the formula (I) and (II). Preferredembodiments relate to pharmaceutically acceptable derivatives ofcompounds of formulas (I) and (II) that are hydrates.

Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic and organic acids and bases.Examples of suitable acids include hydrochloric, hydrobromic, sulphuric,nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic,salicylic, succinic, toluene-p-sulfuric, tartaric, acetic, citric,methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfuric andbenzenesulfonic acids. Other acids, such as oxalic acid, while notthemselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsand their pharmaceutically acceptable acid addition salts. Salts derivedfrom appropriate bases include alkali metal (e.g., sodium), alkalineearth metal (e.g. magnesium), ammonium and N—(C₁-C₄alkyl)₄ ⁺ salts.

In addition, the scope of the invention also encompasses prodrugs ofcompounds of the formulas (I) and (II). Prodrugs include those compoundsthat, upon simple chemical transformation, are modified to producecompounds of the invention. Simple chemical transformations includehydrolysis, oxidation and reduction. Specifically, when a prodrug isadministered to a patient, the prodrug may be transformed into acompound disclosed hereinabove, thereby imparting the desiredpharmacological effect.

The compounds of the invention have demonstrated a selective inhibitionof the bacterial ribosome. These drugs do not affect the eukaryoticribosome because they do not target eukaryotic mitochondrial orcytosolic 16S ribosomal RNA as demonstrated in tests with geneticallyengineered ribosomes carrying eukaryotic 16 S RNA nucleotide positions.

Hence, in a further aspect the present invention is directed to the useof one or more compounds according to the invention for preparing amedicament. Preferably, the compounds of the invention are used forpreparing a medicament for the treatment and/or prevention of abacterial infection.

In the above respect the present invention also relates to apharmaceutical composition, comprising as active substance one or morecompounds according to the invention or pharmaceutically acceptablederivatives or prodrugs thereof, optionally combined with conventionalexcipients and/or carriers.

Methods of Use

For therapeutic or prophylactic use the compounds of the invention maybe administered in any conventional dosage form in any conventionalmanner. Routes of administration include, but are not limited to,intravenously, intramuscularly, subcutaneously, intrasynovially, byinfusion, sublingually, transdermally, orally, topically, or byinhalation. The preferred modes of administration are oral andintravenous.

The compounds may be administered alone or in combination with adjuvantsthat enhance stability of the inhibitors, facilitate administration ofpharmaceutical compositions containing them in certain embodiments,provide increased dissolution or dispersion, increase inhibitoryactivity, provide adjunct therapy, and the like, including other activeingredients. Advantageously such combination therapies utilize lowerdosages of the conventional therapeutics, thus avoiding possibletoxicity and adverse side effects incurred when those agents are used asmonotherapies. The above described compounds may be physically combinedwith the conventional therapeutics or other adjuvants into a singlepharmaceutical composition. Reference is this regard may be made toCappola et al.: U.S. patent application Ser. No. 09/902,822, PCT/US01/21860 and U.S. provisional application No. 60/313,527, eachincorporated by reference herein in their entirety. Advantageously, thecompounds may then be administered together in a single dosage form. Insome embodiments, the pharmaceutical compositions comprising suchcombinations of compounds contain at least about 5%, but more preferablyat least about 20%, of a compound of formula (2) or (3) (w/w) or acombination thereof. The optimum percentage (w/w) of a compound of theinvention may vary and is within the purview of those skilled in theart. Alternatively, the compounds may be administered separately (eitherserially or in parallel). Separate dosing allows for greater flexibilityin the dosing regime.

As mentioned above, dosage forms of the compounds described hereininclude pharmaceutically acceptable carriers and adjuvants known tothose of ordinary skill in the art. These carriers and adjuvantsinclude, for example, ion exchangers, alumina, aluminium stearate,lecithin, serum proteins, buffer substances, water, salts orelectrolytes and cellulose-based substances. Preferred dosage formsinclude, tablet, capsule, caplet, liquid, solution, suspension,emulsion, lozenges, syrup, reconstitutable powder, granule, suppositoryand transdermal patch. Methods for preparing such dosage forms are known(see, for example, H. C. Ansel and N. G. Popovish, Pharmaceutical DosageForms and Drug Delivery Systems, 5^(th) ed., Lea and Febiger (1990)).Dosage levels and requirements are well-recognized in the art and may beselected by those of ordinary skill in the art from available methodsand techniques suitable for a particular patient. In some embodiments,dosage levels range from about 1-100 mg/dose for a 70 kg patient.Although one dose per day may be sufficient, up to 5 doses per day maybe given. For oral doses, up to 2000 mg/day may be required. Referencein this regard may also be made to U.S. provisional application No.60/339,249. As the skilled artisan will appreciate, lower or higherdoses may be required depending on particular factors. For instance,specific doses and treatment regimens will depend on factors such as thepatient's general health profile, the severity and course of thepatient's disorder or disposition thereto, and the judgment of thetreating physician.

For example, the compounds of the present invention can be administeredthe same way as paromomycin and other paromamine compounds. They may beadministered orally in the treatment of intestinal infections orparenterally for visceral and topically for cutaneous leishmaniasis.

Preferably, the compounds of the invention are administered as sulfatesalts. In the case of intestinal amoebiasis the oral dosage for bothadults and children may be 5 to 70, preferably 15 to 50, more preferably25 to 35 mg compound of the invention per kg body weight daily,administered in three doses with meals for five to ten days. In the caseof hepathic coma, the oral daily dosage is 4 g in divided doses given atregular intervals for 5 to 6 days.

Compounds of the invention may be formulated into capsules the same wayparomomycin is formulated (e.g. Humatin®, Parke-Davis). Each capsule maycontain 100 to 500, preferably 150 to 300, more preferably 200 to 250 mgof a compound of the invention. For example, nonmedicinal ingredients incapsules for the compounds of the present invention are—capsule shell:D&C yellow No. 10, FD&C blue No. 1, FD&C red No. 3, FD&C yellow No. 6,gelatin and titanium dioxide. Bottles of 100. (see also Martindale: thecomplete drug reference, 34^(th) Edition, 2005, Pharmaceutical Press, p612.)

It is emphasized that compounds of the present invention may beadministered in higher dosages than paromomycin or other unspecificantibiotics because the compounds of the invention are selective formicrobial, in particular bacterial, ribosomes and spare eukaryoticribosomes, thus lacking serious side effects.

Methods for Preparing Compounds of the Invention

The compounds of the invention can be prepared by standard methods wellknown to) those of skill in the art, in particular in the art of organicand glycoside chemistry. General schemes and specific examples for theirpreparation are provided for in the examples below which are by no meansconsidered limiting but meant as illustrative only.

In the following a preferred method for preparing compounds of theinvention is provided which is also considered the best mode forpracticing the invention.

In a further aspect the invention relates to a method for preparing acompound according to the invention, comprising one or more of thefollowing steps:

a) providing a compound according to the formula III:

wherein:V and W denote in each case, independently of one another, —O—, —NH— and—S—;R³ and R⁴ denote in each case, independently of one another, hydrogen,amino or hydroxyl;R⁵ and R⁶ denote in each case, independently of one another, hydrogen orglycosyl residues;b) protecting one or more, preferably all, of the amino groups;c) optionally protecting one or more, preferably all, of the hydroxygroups;d1) selectively transforming the optionally protected 4′- and/or6′-hydroxy groups to YR¹ and/or ZR²-groups of formula I or the ringsystem of formula II; ord2) selectively deprotecting the protected 4′- and/or 6′-hydroxy groupsand selectively transforming the deprotected 4′- and/or 6′-hydroxygroups to YR¹ and/or ZR²-groups of formula I or the ring system offormula II;e) deprotecting the one or more amino groups;f) deprotecting the one or more hydroxy groups;wherein the order of steps b) and c) as well as e) and f) may bereversed, the order of b) and c) as well as e) and f) being preferred.

Regarding the option of step c) it is noted that for preparing somecompounds of the invention, e.g. compounds of formula II, it may not benecessary to protect one or more of the hydroxyl groups. This isespecially true when a dioxane, dioxane derivative or otherarylalkylidene (such as benzylidene) moiety is introduced to generate acompound of formula II. In that case the moiety introduced in step d1)may already function as a protecting group.

When V and/or W is a divalent sulfur atom, R¹ and/or R²-groups offormula I are introduced by reaction with an appropriate electrophile,such as a halogen derivative, or a sulfonate. Alternatively, the sameresulting YR¹ and/or ZR² may be introduced, directly or indirectly, froman optionally protected compound of formula III where VH and WH denoteindependently of one another a hydroxy group. The ring system of formulaII may be introduced by acetal formation with R⁷CHO orR⁷CH(OR^(a))(OR^(b)), wherein R^(a) and R^(b) are alkyl residues,preferably methyl, in the presence of a suitable organic and/orinorganic acid and, if necessary, in a suitable solvent, or by reactionwith

wherein X is a leaving group, preferably a halogen atom, more preferablybromine, in the presence of a base and, if necessary, in a suitablesolvent.

It is noted that the protecting and deprotecting steps for amino and/orhydroxyl groups can be accomplished by any suitable reactions that areroutinely available to the skilled person and that are not detrimentalto the compounds of the invention.

Preferably the amino groups are protected by converting them to azido,carbamoyl or N-acylamino groups. More preferably the amino groups areprotected by converting them to azido groups by diazo transfer.

Preferably the hydroxy groups are protected by converting them toethers, preferably alkyl and/or silyl ethers, and/or esters, preferablysulfonates or acetals. More preferred the hydroxy groups are protectedas acetoxy groups by converting them with an acyl transfer reagent,preferably acetic anhydride, preferably in the presence of bases and/orcatalysts.

For the acetoxy groups it is preferred that they are deprotected bybase- or acid-catalyzed hydrolysis, or other solvolysis, or by reductionwith a hydride donor, such as diisobutyl aluminum hydride, LiAlH₄, orLiBH₄.

In another preferred embodiment 4′,6′-acetals of formula III areprepared by reaction of a hydroxy compound of formula III with R⁷CHO orR⁷CH(OR^(a))(OR^(b)), wherein R^(a) and R^(b) are alkyl residues,preferably methyl, in the presence of a suitable organic and/orinorganic acid and, if necessary, in a suitable solvent.

In another preferred embodiment 4′,6′-acetals of formula III areprepared by reaction of a hydroxy compound of formula III with

wherein X is a leaving group, preferably a halogen atom, more preferablybromine, in the presence of a base and, if necessary, in a suitablesolvent.

Azido groups are preferably deprotected by a method selected from thegroup consisting of:

reductive hydrogenation, preferably hydrogen in the presence of asuitable catalyst, such as, e.g. Pd, Pd(OH)₂, Rh, or Pt;by hydrogen and nickel boride;reaction with a hydride reagent, preferably a borohydride or an aluminumhydride, electron transfer, preferably by reaction with hydrogensulfide, a thiol, or a dithiol in the presence of base,a metallic reduction, such as by Zn or Na, or Na-amalgam in the presenceof an alcohol, andtreatment with a phosphine reagent, preferably with trimethylphosphine(Staudinger reaction) followed by hydrolysis (see Staudinger and Meyer,Helv. Chim. Acta 1919, 2, 635; Golobov et al., Tetrahedron, 1981, 37,437; and Scriven et al., Chem. Rev., 1988, 88, 297; and as described inS. D. Burke and R. L. Danheiser, ed. “Oxidizing and Reducing Agents”, J.Wiley, Chichester, 1999).

In a preferred embodiment the present invention relates to a method,wherein 4′- and 6′-hydroxy groups are selectively deprotected.

In another preferred embodiment the 4′- and 6′-hydroxy groups areprotected as a benzylidene acetal. Said 4′,6′-benzylidene acetal ispreferably deprotected by a method selected from the group consisting ofcatalytic hydrogenation, Birch reduction and acid-catalyzed hydrolysis.

In a preferred embodiment the method of the invention comprises theregioselective reductive cleavage of the 4′,6′-acetal to result in themore hindered ether derivative by reaction with a suitable hydridereagent, preferably the borane-dimethyl sulfide complex in the presenceof a suitable Lewis acid.

In another preferred embodiment the method of the invention comprisesthe regioselective reductive cleavage of the 4′,6′-acetal to result inthe less hindered ether derivative by reaction with a suitable hydridereagent, preferably sodium cyanoborohydride in the presence of asuitable acid.

In another preferred embodiment hydroxy groups in the compounds offormula III are protected as benzyloxy groups, the benzyl partpreferably being substituted or unsubstituted arylmethyl orheteroarylmethyl. Most preferred the benzyloxy group is ap-methoxybenzyloxy group, preferably being introduced by reaction of acompound of formula III with a suitable p-methoxybenzyl transfer reagentsuch as a p-methoxybenzyl halide in the presence of a suitable base oracid. In a preferred embodiment the p-methoxybenzyloxy groups aredeprotected by oxidative cleavage with a suitable reagent, preferablywith dichlorodicyanoquinone.

In amore preferred embodiment the method of the invention requires thatthe primary 6′-hydroxy group of formula III is selectively protected asan ether, preferably an alkyl or silyl ether, or an ester, preferably asulfonate or an acetal. It is most preferred that the primary 6′-hydroxygroup is a monomethoxytrityloxy group, preferably introduced intocompounds of formula III by reaction with a suitable monomethoxytrityltransfer reagent such as a monomethoxytrityl halide in the presence of asuitable base.

Last but not least, it is preferred that the 4′-hydroxy group isprotected as an ether, preferably by reaction with a suitable alkoxytransfer reagent, preferably an alkyl halide in the presence of asuitable base.

FIGURES

FIG. 1: illustrates the strategy for deletion of rRNA operon rrnBaccording to example 3. Open arrows represent rRNA genes; P and T thepromoter and termination sequences, respectively. Solid arrows indicatethe open reading frames upstream and downstream of rrnB. Hatchedrectangles represent antibiotic resistance cassettes, the stippled arrowthe sacB gene. Broken lines indicate possible crossover sites betweenhomologous sequences in the replacement vector (A) and the chromosomaltarget site (B). Following plasmid integration into the rrnB 5′-flankingregion (C), a second crossover event) between the homologous 3′-flankingsequences resolves the chromosomal tandem repeat to the deletion of rrnB(D).

FIG. 2 illustrates the sequential strategy for the generation of M.smegmatis ΔrrnA ΔrrnB attB::prrnB according to example 3. Followingdeletion of chromosomal rrnB, a complementation vector carrying afunctional rmB operon is introduced to the chromosomal attB site.Subsequent deletion of rrnA results in strain M. smegmatis ΔrrnA ΔrrnBattB::prrnB, in which ribosomal RNA is exclusively transcribed from theplasmid.

In the following the subject-matter of the invention will be describedin more detail referring to specific embodiments which are not intendedto be construed as limiting to the scope of the invention.

Brauchen wir diese Liste wirklich? Die meisten AbkOrzungen sind demFachmann/Fachfrau geläufig. Ich habe schon mal ein paar gestrichen, diewir mit Sicherheit nicht brauchen.

LIST OF ABBREVIATIONS

ax=axial, c=concentration, DDQ=dichlorodicyanoquinone,dec.=decomposition, eq=equatorial, equ.=equivalent, ESI=electrosprayionisation, FC=flash chromato-graphy, HR=high resolution,HSQC=heteronuclear single quantum coherence, MMTr=monomethoxytrityl, —(XÅ) MS=molecular sieves, PMB=4-methoxybenzyl,Tf=trifluoromethanesulfonyl, Ts=4-toluenesulfonyl.

EXAMPLES Example 1 Materials and Methods

Solvents were freshly distilled: THF from Na and benzophenone, CH₂Cl₂,MeOH, pyridine, Et₃N from CaH₂. Reactions were carried out undernitrogen, unless stated otherwise. Qual. TLC: precoated silica-gel glassplates (Merck silica gel 60 F₂₅₄);) detection by heating with ‘mostain’(400 ml of 10% H₂SO₄ soln., 20 g of (NH₄)₆Mo₇O₂₄.6H₂O, 0.4 g ofCe(SO₄)₂). Flash chromatography (FC): silica gel Merck 60 (0.063-0.20).M.p.'s uncorrected. Optical rotations: 1-dm cell at 25°, 589 nm. FT-IRspectra: ATR, absorption in cm⁻¹. ¹H- and ¹³C-NMR spectra: chemicalshifts δ in ppm rel. to TMS as external standard, and coupling constantsJ in Hz. HR-ES-MS or HR-MALDI-MS: in gentisic acid(=2,5-dihydroxybenzoic acid, DHB) matrix.

Synthesis of Acetals

Compounds 2, 3, 4.1 were synthesized as described in Helv Chim Acta2005, 88, 2967.

Typical procedure for the formation of the acetals 4.2-4.12 (Scheme 1).Under N₂ a soln. of 2 in DMF was treated with a solution of thecorresponding dimethylacetal of R⁷CHO (5 equ.) in DMF and TsOH.H₂O (0.5equ.) and, stirred at 25° or at 65°. The mixture was diluted with AcOEtand washed with 0.1 M aq. NaOH. The aqueous layer was extracted twicewith AcOEt. The combined org. layers were washed with brine, dried(MgSO₄), filtered, and evaporated. FC (Hexane/AcOEt 8:2, CHCl₃/AcOEt1:1→CHCl₃/AcOEt/CH₃OH 6:6:0.25) gave 4.2-4.12.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-p-Methoxybenzylideneparomomycin(4.2). Reaction of 71 mg of 2 gave 57 mg of 4.2 (69%). White solid.R_(f) (CHCl₃/AcOEt/MeOH 3:3:0.5) 0.46. [α]_(D) ²⁹=+112.5 (c=0.29,CH₃OH). HR-Maldi-MS: 902.2569 (22, [M+K]⁺, C₃₁H₄₁N₁₅O₁₅ ⁺; calc.902.2544); 887.2817 (39, [M+Na+H]⁺, C₃₁H₄₂N₁₅NaO₁₅ ⁺; calc. 887.2883);886.2783 (100, [M+Na]⁺, C₃₁H₄₁N₁₅NaO₁₅ ⁺; calc. 886.2799). Anal. calc.for C₃₁H₄₁N₁₅O₁₅.CH₃OH (895.79): C, 42.91; H, 5.06; N, 23.45; found: C,43.31; H, 5.00; N, 23.14.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-m-methoxybenzylideneparomomycin(4.3). Reaction of 101 mg of 2 gave 47 mg of 4.3 (40%). White solid.R_(f) (CHCl₃/AcOEt/MeOH 3:3:0.5) 0.45. [α]_(D) ²⁹=+98.2 (c=0.17, CH₃OH).HR-Maldi-MS: 863.2932 (33, [M]⁺, C₃₁H₄₁N₁₅O₁₅ ⁺; calc. 863.2907);862.2849 (88, [M−H]⁺, C₃₁H₄₀N₁₅O₁₅ ⁺; calc. 862.2834). Anal. calc. forC₃₁H₄₁N₁₅O₁₅.CH₃CO₂CH₂CH₃ (951.85): C, 44.16; H, 5.19; N, 22.07; found:C, 43.83, H, 5.32, N, 21.71.

1,3,2′,2′″,6′″-Pentadeamino-4,3,2′,2″,6′″-pentaazido-4,6′-O-o-methoxybenzylideneparomomycin(4.4). Reaction of 99 mg of 2 gave 76 mg of 4.4 (66%). White solid.R_(f) (CHCl₃/AcOEt/MeOH 3:3:0.5) 0.40. [α]_(D) ²⁹=+120.6 (c=0.28,CH₃OH). HR-Maldi-MS: 887.2812 (33, [M+Na+H]⁺, C₃₁H₄₃N₁₅NaO₁₅ ⁺; calc.887.2883); 886.2784 (100, [M+Na]⁺, C₃₁H₄₂N₁₅NaO₁₅ ⁺; calc. 886.2799).Anal. calc. for C₃₁H₄₁N₁₅O₁₅.CH₃OH (895.79): C, 42.91; H, 5.06; N,23.45; found: C, 43.01; H, 5.23; N, 23.37.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-2,5-dimethoxybenzylideneparomomycin(4.5). Reaction of 119 mg of 2 gave 104 mg of 4.5 (73%). White solid.R_(f)(CHCl₃/AcOEt/MeOH 3:3:0.5) 0.54. [α]_(D) ²⁹=+105.0 (c=0.295,CH₃OH). HR-Maldi-MS: 917.2917 (40, [M+Na+H]⁺, C₃₂H₄₄N₁₅NaO₁₆ ⁺; calc.917.2988); 916.2887 (100, [M+Na]⁺, C₃₂H₄₃N₁₅NaO₁₆ ⁺; calc. 916.2904).Anal. calc. for C₃₂H₄₃N₁₅O₁₆.CH₃OH (925.82): C, 42.81; H, 5.12; N,22.69; found: C, 42.93; H, 5.34; N, 22.34.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2″,6′″-pentaazido-4′,6′-O-p-nitrobenzylideneparomomycin(4.6). Reaction of 49 mg of 2 gave 104 mg 4.6 (35%). White solid. R_(f)(CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.36. HR-Maldi-MS: 878.2619 (39, [M]⁺,C₃₀H₃₈N₁₆O₁₆ ⁺; calc. 878.2652); 877.2570 (100, [M−H]⁺, C₃₀H₃₇N₁₆O₁₆ ⁺;calc. 877.2573).

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,6=0-m-nitrobenzylideneparomomycin(4.7). Reaction of 100 mg of 2 gave 29 mg of 4.7 (26%). White solid.R_(f) (CHCl₃/AcOEt/MeOH 3:3:0.5) 0.36. HR-Maldi-MS: 878.2604) (44, [M]⁺,C₃₀H₃₈N₁₆O₁₆ ⁺; calc. 878.2652); 877.2561 (100, [M−H]⁺, C₃₀H₃₇N₁₆O₁₆ ⁺;calc. 877.2573).

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-p-chlorobenzylideneparomomycin(4.8). Reaction of 28 mg of 2 gave 10 mg 4.8 (31%).R_(f)(CHCl₃/AcOEt/CH₃OH 3:3:1) 0.48.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,dichlorobenzylideneparomomycin (4.9). Reaction of 80 mg of 2 gave 22 mgof 4.7 (23%). White solid. R_(f) (CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.39.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-p-cyanobenzylideneparomomycin(4.10). Reaction of 115 mg of 2 gave 43 mg of 4.10 (33%). White solid.R_(f) (CHCl₃/AcOEt/MeOH 3:3:0.5) 0.31. HR-Maldi-MS: 858.2722 (39, [M]⁺,C₃₁H₃₈N₁₆O₁₄ ⁺; calc. 858.2753); 857.2675 (100, [M−H]⁺, C₃₁H₃₇N₁₆O₁₄ ⁺;calc. 857.2681).

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-p-phenylbenzylideneparomomycin(4.11). Reaction of 80 mg of 2 gave 66 mg of 4.11 (68%). White solid.R_(f)(CHCl₃/AcOEt/MeOH 3:3:0.5) 0.42. [α]_(D) ²⁹=+102.6 (c=0.29, CH₃OH).HR-Maldi-MS: 908.3058 (100, [M−H]⁺, C₃₆H₄₂N₁₅O₁₄ ⁺; calc. 908.3041);909.3147 (40, [M]⁺, C₃₆H₄₃N₁₅O₁₄ ⁺; calc. 909.3114). Anal. calc. forC₃₆H₄₃N₁₅O₁₄.CH₃OH (941.34): C, 47.18; H, 5.03; N, 22.31; found: C,47.26, H, 5.11, N, 22.39.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-p-fluorobenzylideneparomomycin(4.12). Reaction of 175 mg of 2 gave 139 mg of 4.12 (69%). White solid.R_(f) (CHCl₃/AcOEt/MeOH 3:3:0.5) 0.34. HR-Maldi-MS: 890.2335 (28,[M+K]⁺, C₃₀H₃₈FKN₁₅O₁₄ ⁺; calc. 890.2344); 875.2620 (38, [M+Na+H]⁺,C₃₀H₃₉FN₁₅NaO₁₄ ⁺; calc. 875.2683); 874.2582 (100, [M+Na]⁺,C₃₀H₃₈FN₁₅NaO₁₄ ⁺; calc. 874.2599). Anal. calc. for C₃₀H₃₈FN₁₅O₁₄.CH₃OH(883.7551): C, 42.13; H, 4.79; N, 23.77; found: C, 42.39; H, 4.79; N,23.77.

Typical procedure for the deprotection of azido groups. Under N₂, asoln. of 4.1-4.12 in THF was treated with 0.1 M aq. NaOH (2 equ.) and 1MPMe₃ in THF (6 equ.), stirred at 60°. Evaporation and FC (THF, THF/MeOH,MeOH, MeOH/25% aq. NH₃ 49:1→MeOH/25% aq. NH₃ 4:1) gave 5.1-5.12.

4′,6′-O-benzylideneparomomycin (5.1). Reaction of 110 mg of 4.1 gave 73mg of 5.1 (79%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1) 0.30. [α]_(D)²⁵=+43.2 (c=0.25, H₂O). IR (ATR): 3355w, 3290w, 2909w, 1589w, 1455w,1378w, 1334w, 1023s, 978s, 927m. ¹H-NMR (500 MHz, D₂O, assignment basedon DQF COSY and a HSQC spectrum): 7.57-7.55 (m, 2 arom. H); 7.50-7.48(m, 3 arom. H); 5.58 (s, PhCH); 5.55 (d, J=3.8, H—C(1′)); 5.40 (d,J=2.3, H—C(1″)); 5.04 (d, J=1.7, H—C(1′″)); 4.44 (dd, J=6.6, 5.0,H—C(3″)); 4.35-4.31 (m, 2H, H—C(2″), F1, —C(6′)); 4.18-4.13 (m, 2H,H—C(4″), H—C(5′″)); 4.10-4.04 (m, 2H, H—C(3″), H—C(5′)); 3.94-3.87 (m,2H, H_(a) C(5″), H—C(3′)); 3.91 (t, J=10.5, H_(b)—C(6′)); 3.77-3.73 (m,2H, H—C(4′), H—C(5)); 3.74 (dd, J=10.0, 5.2, H_(b)—C(5″)); 3.71-3.70 (m,1H, H—C(4′″)); 3.54 (t, J=9.3, H—C(4)); 3.40 (t, J=9.7, H—C(6); 3.28(dd, J=13.6, 7.9, H_(a)—C(6′″)); 3.21 (dd, J=13.5, 3.9, H_(b)—C(6′″));3.13 (m, 1H, H—C(2″)); 3.04-3.00 (m, 1H, H—C(3)); 2.99 (dd, J=10.0, 3.9,H—C(2′)); 2.94-2.89 (m, 1H, H—C(1)); 2.07 (dt, J=13.0, 4.1,H_(eq)—C(2)); 1.34 (q, J=12.5, H_(ax)—C(2)). ¹³C-NMR (126 MHz, D₂O,assignment based on a HSQC spectrum): 138.79 (s); 132.61 (d), 131.43(2d); 128.91 (2d); 111.35 (d, C(1″)); 104.52 (d, PhCH); 101.83 (d,C(1′)); 101.18 (d, C(1′″)); 86.96 (d, C(5)); 84.46 (d, C(4)); 83.89 (d,C(4″)); 83.52 (d, C(4′)); 78.88 (d, C(6)); 78.06 (d, C(3″)); 76.05 (d,C(2″)); 74.61 (d, C(5′″)); 72.73 (d, C(3′″)); 72.56 (d, C(3′), 71.11 (d,C(4′″)); 70.72 (t, C(6′)); 66.32 (d, C(5′)); 63.38 (t, C(5″)); 58.55 (d,C(2′)); 54.87 (d, C(2″); 53.02 (d, C(1)); 52.24 (d, C(3)); 43.41 (t,C(6′″)); 36.81 (t, C(2)). HR-Maldi-MS: 726.3117 (31, [M+Na]⁺,C₃₀H₄₉N₅NaO₁₄ ⁺; calc. 726.3174); 705.3347 (37, [M+2H]⁺, C₃₀H₅₁N₅O₁₄ ⁺;calc. 705.3433); 704.3336 (100, [M+H]⁺, C₃₀H₅₀N₅O₁₄ ⁺; calc. 704.3349).Anal. calc. for C₃₀H₄₉N₅O₁₄ (703.74): C, 51.20; H, 7.02; N, 9.95; found:C, 51.30; H, 7.15; N, 9.98.

4′,6′-O-p-Methoxybenzylideneparomomycin (5.2). Reaction of 17 mg of 4.2gave 11 mg of 5.2 (76%). White solid.

R_(f) (MeOH/25% aq. NH₃ 4:1) 0.36. IR (ATR): 3355w (br.), 2917w, 1614w,1518w, 1461w, 1378w, 1302w, 1250w, 1101s, 1024s, 937m. HR-Maldi-MS:735.3475 (36, [M+2H]⁺, C₃₁H₅₃N₅O₁₅ ⁺; calc. 735.3538); 734.3442 (100,[M+H]⁺, C₃₁H₅₂N₅O₁₅ ⁺; calc. 734.3454).

4′,6′-O-m-Methoxybenzylideneparomomycin tetraacetate (5.3). Reaction of26 mg of 4.3 gave 18 mg of 5.3 (81%). White solid. The product wasconverted into the tetraacetate salt by stirring in 20% aq. AcOH (23mg). R_(f) (MeOH/25% aq. NH₃ 4:1) 0.34. IR (ATR): 3311w, 2884w, 1542m,1402m, 1336w, 1285w, 1261w, 1096s, 1042s, 1014s. HR-Maldi-MS: 757.3323(28, [M+Na+H]⁺, C₃₁H₅₂N₅NaO₁₅ ⁺; calc. 757.3358); 756.3287 (83, [M+Na]⁺,C₃₁H₅₁N₅NaO₁₅ ⁺; calc. 756.3279); 735.3468 (37, [M+2H]⁺, C₃₁H₅₃N₅O₁₅ ⁺;calc. 735.3538); 734.3442 (100, [M+H]⁺, C₃₁H₅₂N₅O₁₅ ⁺; calc. 734.3454);574.2595 (31, [M−ring IV+2H]⁺, C₂₅H₄₀N₃O₁₂ ⁺; calc. 574.2612).

4,6′-O-o-Methoxybenzylideneparomomycin (5.4). Reaction of 18 mg of 4.4gave 21 mg of 5.4. White solid. R_(f) (MeOH/25% aq. NH₃ 4:1) 0.31.[α]_(D) ²⁵=+51.3 (c=0.15, H₂O). IR (ATR): 3148w (br.), 1606w, 1526w,1498w, 1463w, 1386w, 1287w, 1250w, 1095s, 1047s. HR-Maldi-MS: 757.3328(25, [M+Na+H]⁺, C₃₁H₅₂N₅NaO₁₅ ⁺; calc. 757.3358); 756.3291 (67, [M+Na]⁺,C₃₁H₅₁N₅NaO₁₅ ⁺; calc. 756.3279); 735.3470 (36, [M+2H]⁺, C₃₁H₅₃N₅O₁₅ ⁺;calc. 735.3538); 734.3442 (100, [M+H]⁺, C₃₁H₅₂N₅O₁₅ ⁺; calc. 734.3454);442.15.17 (32, [M−ring III−ring IV+2 H]⁺, C₂₀H₃₂N₃O₈ ⁺; calc. 442.2189).

4′,6′-O-2,5-Dimethoxybenzylideneparomomycin (5.5). Reaction of 25 mg of4.5 gave 21 mg of 5.5 (98%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.29. IR (ATR): 3287w, 2913w, 1593w, 1503m, 1464m, 1425w, 0.1386w,1280w, 1221w, 1104s, 1019s. HR-Maldi-MS: 786.3382 (26, [M+Na]⁺,C₃₂H₅₃N₅NaO₁₆ ⁺; calc. 786.3385); 765.3571 (37, [M+2H]⁺, C₃₂H₅₅N₅O₁₆ ⁺;calc. 765.3644); 764.3545 (100, [M+H]⁺, C₃₂H₅₄N₅O₁₆ ⁺; calc. 764.3560).

4′,6′-O-p-Nitrobenzylideneparomomycin (5.6). Reaction of 14 mg of 4.6gave 6 mg of 5.6 (50%). White solid. R_(f) (MeOH 25% aq. NH₃ 4:1) 0.24.[α]_(D) ²⁵=+168.1 (c=0.01, H₂O). IR (ATR): 3172w (br.), 2900w, 1611w,1521w, 1349w, 1130s, 1099s, 995s. HR-Maldi-MS: 750.2943 (38, [M+2H]⁺,C₃₀H₅₀N₆O₁₆ ⁺; calc. 750.3283); 749.3196 (100, [M+H]⁺, C₃₀H₄₉N₆O₁₆ ⁺;calc. 749.3205).

4,6′-O-m-Nitrobenzylideneparomomycin triacetate (5.7). Reaction of 16 mgof 4.7 gave after stirring in 10% aq. AcOH and evaporation 11 mg of 5.7(61%): Yellowish solid. R_(f) (MeOH/25% aq. NH₃ 4:1) 0.31. IR (ATR):3095m (br.), 1528m, 1406m, 1349m, 1100s, 1045s, 1014s, 937m.HR-Maldi-MS: 750.3211 (36, [M+2H]⁺, C₃₀H₅₀N₆O₁₆ ⁺; calc. 750.3283);749.3187 (100, [M+H]⁺, C₃₀H₄₉N₆O₁₆ ⁺; calc. 749.3200).

4′,6′-O-p-Chlorobenzylideneparomomycin (5.8). Reaction of 17 mg of 4.8gave 12 mg of 5.8 (83%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1) 0.36.IR (ATR): 3037w (br.), 1738w, 1603w 1524w, 1493w, 1454w, 1406w, 1375w,1081s, 1049s, 1018s. HR-Maldi-MS: 738.2972 (95, [M+H]⁺, C₃₀H₄₉ClN₅O₁₄ ⁺;calc. 750.3283).

4,6′-O-3,5-Dichlorobenzylideneparomomycin (5.9). Reaction of 25 mg of4.9 gave 19 mg of 5.9 (89%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.29. IR (ATR): 3188w (br.), 2900w, 1597w, 1573w 1526w, 1429w, 1369w,1101s, 1029s. HR-Maldi-MS: 772.2556 (100, [M+H]⁺, C₃₀H₄₈Cl₂N₅O₁₄ ⁺;calc. 772.2575).

4,6′-O-p-Cyanobenzylideneparomomycin (5.10). Reaction of 10 mg of 4.10gave 9 mg of 5.10. White solid. R_(f) (MeOH/25% aq. NH₃ 4:1) 0.27. IR(ATR): 3200w (br.), 1619w, 1526w, 1417w, 1094s, 996s. HR-Maldi-MS:730.3322 (36, [M+2H]⁺, C₃₁H₅₀N₆O₁₄ ⁺; calc. 730.3385); 729.3288 (100,[M+H]⁺, C₃₁H₄₉N₆O₁₄ ⁺; calc. 729.3307).

4′,6′-O-p-Phenylbenzylideneparomomycin (5.11). Reaction of 20 mg of 4.11gave 16 mg of 5.11 (93%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.31. [α]_(D) ²⁵=+42.3 (c=0.14, H₂O). IR (ATR): 3356w, 3284w, 2903w,1599w, 1532w, 1489w, 1451w, 1375w, 1110s, 1074s, 1026s. HR-Maldi-MS:803.3565 (41, [M+Na+H]⁺, C₃₆H₅₄N₅NaO₁₄ ⁺; calc. 803.3565); 802.3462(100, [M+Na]⁺, C₃₆H₅₃N₅NaO₁₄ ⁺; calc.) 802.3481); 780.3616 (48, [M+H]⁺,C₃₆H₅₄N₅O₁₄ ⁺; calc. 780.3667).

4,6′-O-p-Fluorobenzylideneparomomycin (5.12). Reaction of 60 mg of 4.12gave 51 mg of 5.11. White solid. R_(f) (MeOH/25% aq. NH₃ 4:1) 0.33. IR(ATR): 3285w, 2875w, 1606w, 1514w, 1457w, 1376w, 1300w, 1224w, 1047s,1014s, 935m. ¹H-NMR (500 MHz, D₂O, assignment based on DQF COSY and aHSQC spectrum): 7.54-7.50 (m, 2 arom. H); 7.19-7.13 (m, 2 arom. H); 5.71(s, PhCH); 5.51 (d, J=3.9, H—C(1′)); 5.35 (d, J=2.3, H—C(1″)); 5.05 (d,J=1.8, H—C(1′″)); 4.46 (dd, J=6.6, 4.9, H—C(3″)); 4.32 (dd, J=4.9, 2.3,H—C(2″)); 4.27 (dd, J=10.2, 4.8, H_(a)—C(6′)); 4.18-4.11 (m, 2H,H—C(4″), H—C(5′″)); 4.07 (t, J=3.2, H—C(3′″)); 4.03-3.96 (m, H—C(5′));3.88-3.82 (m, 3H, H_(a)—C(5″), H—C(3′), H_(b)—C(6′)); 3.73-3.63 (m, 4H,H_(b)—C(5″), H—C(5), H—C(4′″), H—C(4′)); 3.51 (t, J=9.4, H—C(4)); 3.44(t, J=10.0, H—C(6); 3.32-3.21 (m, 2H, H_(a)—C(6′″), H_(b)—C(6′″)); 3.18(t, J=1.8, H—C(2′″)); 3.01 (dd, J=9.8, 3.9, H—C(2′)); 3.00-2.93 (m, 2H,H—C(3), H—C(1)); 2.08 (dt, J=12.9, 4.1, H_(aq)—C(2)); 1.37 (q, J=12.5,H_(ax)—C(2)). ¹³C-NMR (126 MHz, D₂O, assignment based on a HSQCspectrum): 165.87 (d, ¹J(C,F)=245.7); 135.04 (d, J=2.9, C_(para));131.09 (d, ³J(C,F)=8.7, 2 C_(meta)); 118.18 (d, ²J(C,F)=21.9, 2C_(ortho)); 111.64 (d, C(1″)); 103.83 (d, PhCH); 101.61 (d, C(1′));100.55 (d, C(1′″)); 86.95 (d, C(5)); 84.34 (d, C(4)); 83.91 (d, C(4″));83.38 (d, C(4′)); 78.06 (d, C(3″)); 77.90 (d, C(6)); 76.02 (d, C(2″));73.97 (d, C(5′″)); 72.30 (d, C(3′″)); 72.20 (d, C(3′), 70.90 (d,C(4′″)); 70.67 (t, C(6′)); 66.33 (d, C(5′)); 63.37 (t, C(5″)); 58.42 (d,C(2′)); 54.61 (d, C(2′″)); 53.12 (d, C(1)); 52.15 (d, C(3)); 43.35 (t,C(6′″)); 35.95 (t, C(2)). HR-Maldi-MS: 744.3074 (32, [M+Na]⁺,C₃₀H₄₈FN₅NaO₁₄ ⁺; calc. 744:3079); 723.3262 (34, [M+2H]⁺, C₃₀H₅₀FN₅O₁₄⁺; calc. 723.3338); 722.3242 (100, [M+H]⁺, C₃₀H₄₉FN₅O₁₄ ⁺; calc.722.3255).

Compound 6 was synthesized as described in Helv. Chim. Acta 2005, 88,2967.

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-paromomycin(7). A soln. of 6 (23 mg, 0.021 mmol) in MeOH (1 ml) was treated withTsOH.H₂O (2 mg, 0.011 mmol), stirred at 25° for 5 h. The mixture wasdiluted with AcOEt (10 ml) and washed with H₂Od (8 ml). The aq. Layerwas extracted twice with AcOEt (2×10 ml). The combined org. layers werewashed with brine (20 ml), dried (MgSO₄), filtered and evaporated. FC(Cyclohexane/AcOEt 8:2→cyclohexane/AcOEt 3:7 gave 7 (13 mg, 61%). Whitesolid. R_(f) (CHCl₃/AcOEt/MeOH 3:3:0.5) 0.57.

Typical Procedure for the Formation of the Acetals 4.13-4.27.

Method A: Under N₂, a soln. of 7 in the aldehyde was treated with FeCl₃and, stirred for 2-24 h at 23°. The mixture was diluted with AcOEt,filtered over Celite and washed with 0.1M aq. NaOH. The aqueous layerwas extracted twice with AcOEt. The combined org. layers were washedwith brine, dried (MgSO₄), filtered, and evaporated. FC (Hexane orCyclohexane/AcOEt 8:2→6:4) gave 8.15, 8.19, 8.21-8.27.

Method B: Under N₂, a soln. of 7 in toluene was treated with 5 Åmolecular sieves, TsOH.H₂O (0.5 equ.), a solution of the aldehyde (5equ.) in toluene and, stirred for 2-24 h at reflux. The mixture wasdiluted with AcOEt, flitered over Celite and washed with 0.1M aq. NaOH:The aqueous layer was extracted twice with AcOEt. The combined org.layers were washed with brine, dried (MgSO₄), filtered, and evaporated.FC (Hexane or Cyclohexane/AcOEt 8:2→6:4) gave 8.13-8.14, 8.16-8.18,8.20.)

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-3,5-Dimethoxybenzylideneparomomycin(8.13). Method B: Reaction of 70 mg of 7 gave 58 mg of 8.13 (72%). Whitesolid. R_(f) (Hexane/AcOEt 1:1) 0.34. HR-Maldi-MS (3-HPA): 1184.3294(43, [M+K]⁺, C₄₄H₅₅KN₁₅O₂₂ ⁺; calc. 1184.3283); 1169.3562 (54,[M+Na+H]⁺, C₄₄H₅₆N₁₅NaO₂₂ ⁺; calc. 1169.3622); 1168.3568 (100, [M+Na]⁺,C₄₄H₅₅N₁₅NaO₂₂ ⁺; calc. 1168.3544).

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-3,4,5-trimethoxybenzylideneparomomycin(8.14). Method B: Reaction of 61 mg of 7 gave 50 mg of 8.14 (70%). Whitesolid. R_(f) (Hexane/AcOEt 1:1) 0.34. M.p. 95° (softens)-110°. [α]_(D)²⁵=+92.5 (c=0.23, CHCl₃). HR-Maldi-MS (3-HPA): 1215.3367 (31, [M+K+H]⁺,C₄₅H₅₈KN₁₅O₂₃ ⁺; calc. 1215.3467); 1214.3355) (57, [M+K]⁺, C₄₅H₅₇KN₁₅O₂₃⁺; calc. 1214.3389); 1199.3654 (56, [M+Na+H]⁺, C₄₅H₅₈N₁₅NaO₂₃ ⁺; calc.1199.3728); 1198.3634 (100, [M+Na]⁺, C₄₅H₅₇N₁₅NaO₂₃ ⁺; calc. 1198.3649).Anal. calc. for C₄₅H₅₇N₁₅O₂₃ (1176.03): C, 45.96; H, 4.89; N, 17.87;found: C, 45.87; H, 5.03; N, 17.83.

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-m-chlorobenzylideneparomomycin(8.15). Method A: Reaction of 153 mg of 7 gave 104 mg of 8.15 (61%).White solid. M.p. 76° (softens)-95°. R_(f) (Hexane/AcOEt 1:1) 0.60.[α]_(D) ²⁵=+99.4 (c=0.20, CHCl₃). HR-Maldi-MS (3-HPA): 1158.2693 (35,[M+K]⁺, C₄₂H₅₀ClKN₁₅O₂₀ ⁺; calc. 111158.2682); 1144.2897 (44,[M+Na+2H]⁺, C₄₂H₅₂ClN₁₅NaO₂₀ ⁺; calc. 1144.3099); 1143.2983 (46,[M+Na+H]⁺, C₄₂H₅₁ClN₁₅NaO₂₀ ⁺; calc. 1143.3021); 1142.2934 (100,[M+Na]⁺, C₄₂H₅₀ClN₁₅NaO₂₀ ⁺; calc. 1142.2943).

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-o-nitrobenzylideneparomomycin(8.16). Method B: Reaction of 91 mg of 7 gave 30 mg of 8.16 (29%).

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-p-trifluoromethylbenzylideneparomomycin(8.17). Method B: Reaction of 89 mg of 7 gave 75 mg of 8.17 (73%). Whitesolid. M.p. 71° (softens)-115°. R_(f) (Hexane/AcOEt 1:1) 0.58. M.p.71-105°. [α]_(D) ²⁶=+98.2 (c=0.15, CHCl₃). HR-Maldi-MS (3-HPA):1192.2894 (50, [M+K]⁺, C₄₃H₅₀F₃KN₁₅O₂₀ ⁺; calc. 1192.2946); 1177.3200(52, [M+Na+H]⁺, C₄₃H₅₁F₃N₁₅NaO₂₀ ⁺; calc. 1177.3285); 1176.3182 (100,[M+Na]⁺, C₄₃H₅₀F₃N₁₅NaO₂₀ ⁺; calc. 1176.3201). Anal. calc. forC₄₃H₅₀F₃N₁₅O₂₀.0.5CH₃CO₂CH₂CH₃ (1197.99): C, 45.12; H, 4.54; N, 17.54;found: C, 44.90, H, 4.63, N, 17.29.

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,6′-O-p-dimethylaminobenzylideneparomomycin(8.18). Method B: Reaction of 38 mg of 7 gave 75 mg of 8.18 (42%).Yellowish solid. R_(f) (Cyclohexane/AcOEt 1:1) 0.48. HR-Maldi-MS(3-HPA): 1168.3509 (48, [M+K+H]⁺, C₄₄H₅₇KN₁₆O₂₀ ⁺; calc. 1168.3572);1167.3484 (86, [M+K]⁺, C₄₄H₅₇N₁₆O₂₀ ⁺; calc. 1167.3494); 1152.3798 (47,[M+Na+H]⁺, C₄₄H₅₇N₁₆NaO₂₀ ⁺; calc. 1152.3833); 1151.3769 (89, [M+Na]⁺,C₄₄H₅₆N₁₆NaO₂₀ ⁺; calc. 1151.3754); 1143.4066 (100, [M−CH₃+Na+7H]⁺,C₄₃H₆₀N₁₆NaO₂₀ ⁺; calc. 1143.4067); 1130.3905 (46, [M+2H]⁺, C₄₄H₅₈N₁₆O₂₀⁺; calc. 1130.4013); 1129.3858 (95, [M+H]⁺, C₄₄H₅₇N₁₆O₂₀ ⁺; calc.)1129.3935).

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-1-naphthylideneparomomycin(8.19). Method A: Reaction of 195 mg of 7 gave 157 mg of 8.19 (71%).White solid. R_(f) (Hexane/AcOEt 1:1) 0.55. M.p. 77° (softens)-115°.[α]_(D) ²⁵=+115.5 (c=0.21, CHCl₃). HR-Maldi-MS (3-HPA): 1175.3224 (46,[M+K+H]⁺, C₄₆H₅₄KN₁₅O₂₀ ⁺; calc. 1175.3307); 1174.3203 (82, [M+K]⁺,C₄₆H₅₃KN₁₅O₂₀ ⁺; calc. 1174.3228); 1159.3523 (56, [M+Na+H]⁺,C₄₆H₅₄N₁₅NaO₂₀ ⁺; calc. 1159.3567); 1158.3489 (100, [M+Na]⁺,C₄₆H₅₃N₁₅NaO₂₀ ⁺; calc. 1158.3489). Anal. calc. forC₄₆H₅₃N₁₅O₂₀.0.2CH₃CO₂CH₂CH₃ (1153.62): C, 48.72, H, 4.77, N, 18.21;found: C, 48.76; H, 4.89; N, 17.77.

6,3′,2″,5″,3″,4″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-2-naphthylideneparomomycin(8.20). Method B: Reaction of 80 mg of 7 gave 60 mg of 8.20 (66%). R_(f)(Hexane/AcOEt 1:1) 0.64.

6,3′,2″,5″,3′,4″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,6′-O-2-furanylideneparomomycin(8.21). Method A: Reaction of 146 mg of 7 gave 91 mg of 8.21 (58%). Paleorange solid. R_(f) (Hexane/AcOEt 1:1) 0.60. M.p. 98° (softens)-105°.[α]_(D) ²⁵=+117.2 (c=0.11, CHCl₃). HR-Maldi-MS (3-HPA): 1115.2929 (40,[M+K+H]⁺, C₄₀H₅₀KN₁₅O₂₁ ⁺; calc. 1115.2943); 1114.2916 (75, [M+K]⁺,C₄₀H₄₉KN₁₅O₂₁ ⁺; calc. 1114.2865); 1099.3154 (50, [M+Na+H]⁺,C₄₀H₅₀N₁₅NaO₂₁ ⁺; calc. 1099.3203); 1098.3129 (100, [M+Na]⁺,C₄₀H₄₃N₁₅NaO₂₁ ⁺; calc. 1098.3125). Anal. calc. for C₄₀H₄₉N₁₅O₂₁(1075.91): C, 44.65; H, 4.59; N, 19.53; found: C, 44.74; H, 4.59; N,19.05.

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,6′-O-2-thiophenylideneparomomycin(8.22). Method A: Reaction of 80 mg of 7 gave 37 mg of 8.22 (42%). Whitesolid. HR-Maldi-MS (3-HPA): 1130.2660 (52, [M+K]⁺, C₄₀H₄₉KN₁₅O₂₀S⁺;calc. 1130.2636); 1115.2904 (50, [M+Na+H]⁺, C₄₀H₅₀N₁₅NaO₂₀S⁺; calc.1115.2975); 1114.2864 (100, [M+Na]⁺, C₄₀H₄₉N₁₅NaO₂₀S⁺; calc. 1114.2897).Anal. calc. for C₄₀H₄₉N₁₅O₂₀S (1091.98): C, 44.00; H, 4.52; N, 19.24;found: C, 43.86; H, 4.61; N, 19.23.

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-ethylideneparomomycin(8.23). Method A: Reaction of 21 mg of 7 gave 18 mg of 8.23 (84%). Whitesolid. R_(f) (Hexane/AcOEt 1:1) 0.48. HR-Maldi-MS (3-HPA): 1047.3167(46, [M+Na+H]⁺, C₃₇H₅₀N₁₅NaO₂₀ ⁺; calc. 1047.3254); 1046.3153 (100,[M+Na]⁺, C₃₇H₄₉N₁₅NaO₂₀ ⁺; calc. 1046.3176).

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,6′O-(2-phenyl)-ethylideneparomomycin(8.24). Method A: Reaction of 116 mg of 7 gave 97 mg of 8.24 (76%).White solid. M.p. 90° (softens)-96°. R_(f) (Hexane/AcOEt 1:1) 0.46.[α]_(D) ²⁵=+90.0 (c=0.105, CHCl₃). HR-Maldi-MS (3-HPA): 1138.3234 (39,[M+K]⁺, C₄₃H₅₃KN₁₅O₂₀ ⁺; calc. 1138.3228); 1123.3511 (53, [M+Na+H]⁺,C₄₃H₅₄N₁₅NaO₂₀ ⁺; calc. 1123.3567); 1122.3475 (100, [M+Na]⁺,C₄₃H₅₃N₁₅NaO₂₀ ⁺; calc. 1122.3489).

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-(3-phenyl)-propylideneparomomycin(8.25). Method A: Reaction of 177 mg of 7 gave 189 mg of 8.25 (96%).White solid. R_(f) (Hexane/AcOEt 1:1) 0.50. M.p. 66° (softens)-86°.[α]_(D) ²⁵=+108.7 (c=0.24, CHCl₃). HR-Maldi-MS (3-HPA): 1153.3457 (27,[M+K+H]⁺, C₄₄H₅₆KN₁₅O₂₀ ⁺; calc. 1153.3463); 1152.3403 (49, [M+K]⁺,C₄₄H₅₅KN₁₅O₂₀ ⁺; calc. 1152.3385); 1137.3650 (54, [M+Na+H]⁺,C₄₄H₅₆N₁₅NaO₂₀ ⁺; calc. 1137.3724); 1136.3619 (100, [M+Na]⁺,C₄₄H₅₅N₁₅NaO₂₀ ⁺; calc. 1136.3640).

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,6′-O-(3-phenyl)-propenylideneparomomycin(8.26). Method A: Reaction of 75 mg of 7 gave 37 mg of 8.26 (44%).

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,6′-O-cyclohexylmethylideneparomomycin(8.27). Method A: Reaction of 157 mg of 7 gave 83 mg of 8.27 (49%).White solid.

Typical procedure for the formation of 4.13-4.27. A soln. of 8.13-8.27in CH₂Cl₂/MeOH ¼ was treated with NaOMe (12 equ.), stirred at 25°. Themixture was quenched with Amberlite IR-120(H⁺), filtered, andevaporated. FC(CHCl₃/AcOEt 1:1→CHCl₃/AcOEt/MeOH) gave 4.13-4.27.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-3,5-dimethoxybenzylideneparomomycin(4.13). Reaction of 19 mg of 8.13 gave 26 mg of 4.13 (98%). White solid.R_(f)(CHCl₃/AcOEt/MeOH 3:3:0.5) 0.21.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-3,4,5-trimethoxybenzylideneparomomycin(4.14). Reaction of 47 mg of 8.14 gave 30 mg of 4.13 (81%). White solid.R_(f)(CHCl₃/AcOEt/MeOH 3:3:0.5) 0.22.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-m-chlorobenzylideneparomomycin(4.15). Reaction of 70 mg of 8.15 gave 54 mg of 4.15 (quant.). Whitesolid. R_(f)(CHCl₃/AcOEt/MeOH 3:3:0.5) 0.45.

1,3,2′,2″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-o-nitrobenzylideneparomomycin(4.16). Reaction of 54 mg of 8.16 gave 26 mg 4.16 (62%). White solid.R_(f) (CHCl₃/AcOEt/MeOH 3:3:0.5) 0.51.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-p-trifluoromethylbenzylideneparomomycin(4.17). Reaction of 57 mg of 8.17 gave 42 mg of 4.17 (94%). White solid.R_(f) (CHCl₃/AcOEt/MeOH 3:3:0.5) 0.44. HR-Maldi-MS (3-HPA): 924.2549(100, [M+Na]⁺, C₃₁H₃₈F₃N₁₅NaO₁₄ ⁺; calc. 924.2567).

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,6′-O-p-dimethylaminobenzylideneparomomycin(4.18). Reaction of 39 mg of 8.18 gave 22 mg of 4.18 (73%). White solid.R_(f)(CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.21.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-1-naphthylideneparomomycin(4.19). Reaction of 107 mg of 8.19 gave 75 mg of 4.19 (90%). Whitesolid. R_(f) (CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.52.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-2-naphthylideneparomomycin(4.20). Reaction of 60 mg of 8.20 gave 30 mg of 4.20 (65%). White solid.R_(f) (CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.50. ESI-MS: 906.0 (100, [M+Na]⁺,C₃₄H₄₁N₁₅NaO₁₄ ⁺; calc. 906.3). Anal. calc. for C₃₄H₄₁N₁₅O₁₄ (883.79):C, 46.21; H, 4.68; N, 23.77; found: C, 46.44; H, 4.54; N, 23.37.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,6′-O-2-furanylideneparomomycin(4.21). Reaction of 73 mg of 8.21 gave 48 mg of 4.21 (86%). White solid.R_(f)(CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.38.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4,6′-O-2-thiophenylideneparomomycin(4.22). Reaction of 60 mg of 8.22 gave 37 mg of 4.22 (80%). White solid.R_(f) (CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.40.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-ethylideneparomomycin(4.23). Reaction of 29 mg of 8.23 gave 20 mg of 4.23 (92%). White solid.R_(f) (CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.24.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-(2-phenyl)-ethylideneparomomycin(4.24). Reaction of 79 mg of 8.24 gave 55 mg of 4.24 (90%). White solid.R_(f) (CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.46.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-(3-phenyl)-propylideneparomomycin(4.25). Reaction of 127 mg of 8.25 gave 75 mg of 4.25 (76%). Whitesolid. R_(f) (CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.45.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-(3-phenyl)-propenylideneparomomycin(4.26). Reaction of 41 mg of 8.26 gave 24 mg of 4.26 (76%). White solid.R_(f)(CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.48.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-cyclohexylmethylideneparomomycin(4.27). Reaction of 46 mg of 8.27 gave 26 mg of 4.27 (74%). White solid.R_(f) (CHCl₃/AcOEt/CH₃OH 3:3:0.5) 0.37.

Typical procedure for the final deprotection. Under N₂, a soln. of4.13-4.27 in THF was treated with 0.1M aq. NaOH (2 equ.) and 1M PMe₃ inTHF (6 equ.), stirred at 60°. Evaporation and FC (THF, THF/MeOH, MeOH,MeOH/25% aq. NH₃ 49:1→MeOH/25% aq. NH₃ 4:1) gave 5.13-5.27.

4′,6′-O-3,5-Dimethoxybenzylideneparomomycin (5.13). Reaction of 24 mg of4.13 gave 19 mg of 5.13 (92%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.34. IR (ATR): 3179w (br.), 2896w, 1597m, 1463w, 1431w, 1384w, 1338w,1304w, 1200w, 1150s, 1087s, 1047s. HR-Maldi-MS: 765.3554 (36, [M+2H]⁺,C₃₂H₅₅N₅O₁₆ ⁺; calc. 765.3644); 764.3546 (100, [M+H]⁺, C₃₂H₅₄N₅O₁₆ ⁺;calc. 764.3560).

4′,6′-O-3,4,5-Trimethoxybenzylideneparomomycin (5.14). Reaction of 30 mgof 4.14 gave 21 mg of 5.14 (82%). White solid. R_(f) (MeOH/25% aq. NH₃4:1) 0.19. IR (ATR): 3352w, 3289w, 3182w (br.), 2918w, 1593w, 1506w,1462w, 1421w, 1380w, 1330w, 1236w, 1122s, 1026s, 994s.

HR-Maldi-MS: 795.3686 (40, [M+2H]⁺, C₃₃H₅₇N₅O₁₇ ⁺; calc. 795.3749);794.3651 (100, [M+H]⁺, C₃₃H₅₆N₅O₁₇ ⁺; calc. 794.3651).

4′,6′-O-m-chlorobenzylideneparomomycin (5.15). Reaction of 52 mg of 4.15gave 46 mg of 5.15. White solid. R_(f) (MeOH/25% aq. NH₃ 4:1) 0.33.HR-Maldi-MS: 740.2943 (43, [M+3H]⁺, C₃₀H₅₁ClN₅O₁₄ ⁺; calc. 740.3121);739.2982 (38, [M+2H]⁺, C₃₀H₅₀ClN₅O₁₄ ⁺; calc. 739.3043); 738.2945 (100,[M+H]⁺, C₃₀H₄₉ClN₅O₁₄ ⁺; calc. 738.2959).

4′,6′-O-o-Alitrobenzylideneparomomycin (5.16). Reaction of 20 mg of 4.16gave 17 mg of 5.16 (quant.). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.22. IR (ATR): 3179w (br.), 2895w, 1610w, 1528m, 1452w, 1349s, 1099s,1048s, 1028s. HR-Maldi-MS: 771.2961 (31, [M+Na]⁺, C₃₀H₄₈N₆NaO₁₆ ⁺; calc.771.3024); 750.3213 (46, [M+2H]⁺, C₃₀H₅₀N₆O₁₆ ⁺; calc. 750.3283);749.3186 (100, [M+H]⁺, C₃₀H₄₉N₆O₁₆ ⁺; calc. 749.3205).

4′,6′-O-p-Trifluoromethylbenzylideneparomomycin (5.17). Reaction of 31mg of 4.17 gave 26 mg of 5.17 (98%). White solid. R_(f) (MeOH/25% aq.NH₃ 4:1) 0.32. IR (ATR): 3354w (br.), 2921w, 1583w, 1444w, 1378w, 1325s,1122s, 1083s, 1066s, 1017s, 927m. HR-Maldi-MS: 773.3235 (36, [M+2H]⁺,C₃₁H₅₀F₃N₅O₁₄ ⁺; calc. 773.3306); 772.3210 (100, [M+H]⁺, C₃₁H₄₉F₃N₅O₁₄⁺; calc. 772.3223). ¹⁹F NMR: −60.85

4′,6′-O-p-Dimethylaminobenzylideneparomomycin (5.18). Reaction of 16 mgof 4.18 gave 13.5 mg of 5.18 (99%). White solid. R_(f) (MeOH/25% aq. NH₃4:1) 0.27. IR (ATR): 3172w (br.), 2899w, 1615w, 1528w, 1454w, 1378w,1360w, 1100s, 1048s, 996s. HR-Maldi-MS: 748.3811 (38, [M+2H]⁺,C₃₂H₅₆N₆O₁₄ ⁺; calc. 748.3855); 747.3774 (100, [M+H]⁺, C₃₂H₅₅N₆O₁₄ ⁺;calc. 747.3771).

4′,6′-O-1-Naphthylideneparomomycin (5.19). Reaction of 49 mg of 4.19gave 39 mg of 5.19 (93%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.32. IR (ATR): 3345w (br.), 2888w, 1599w, 1532w, 1509w, 1460w, 1392w,1339w, 1272w, 1246w, 1104s, 1054s, 1028s, 996s, 972s, 920s. HR-Maldi-MS:755.3519 (40, [M+2H]⁺, C₃₄H₅₃N₅O₁₄ ⁺; calc. 755.3589); 754.3495 (100,[M+H]⁺, C₃₄H₅₂N₅O₁₄ ⁺; calc. 754.3511).

4′,6′-O-2-Naphthylideneparomomycin (5.20). Reaction of 19 mg of 4.20gave 12 mg of 5.20 (74%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.36. IR (ATR): 3180w (br.), 2895w, 1600w, 1530w, 1467w, 1374w, 1346w,1048s, 1026s HR-Maldi-MS: 776.3309 (56, [M+Na]⁺, C₃₄H₅₁N₅NaO₁₄ ⁺; calc.776.3330); 755.3538 (36, [M+2H]⁺, C₃₄H₅₃N₅O₁₄ ⁺; calc. 755.3589);754.3509 (100, [M+H]⁺, C₃₄H₅₂N₅O₁₄ ⁺; calc. 754.3511); 594.2633 (41,[M−ring IV+2H]⁺, C₂₈H₄₀N₃O₁₁ ⁺; calc. 594.2663); 462.2238 (63, [M−ringIII−ring IV+2H]⁺, C₂₃H₃₂N₃O₇ ^(÷); calc. 462.2240).

4′,6′-O-2-Furanylideneparomomycin (5.21). Reaction of 49 mg of 4.21 gave39 mg of 5.21 (94%). White solid. R_(f) (MeOH/25¹³/0 aq. NH₃ 4:1) 0.21.IR (ATR): 3356w (br.), 2877w, 1664w, 1589w, 1506w, 1460w, 1460w, 1397w,1359w, 1341w, 1135s, 1106s, 1026s, 994s, 918s. HR-Maldi-MS: 716.2997(31, [M+Na]⁺, C₂₈H₄₇N₅NaO₁₅ ⁺; calc. 716.2966); 695.3146 (34, [M+2H]⁺,C₂₈H₄₉N₅O₁₅ ⁺; calc. 695.3225); 694.3128 (100, [M+H]⁺, C₂₈H₄₈N₅O₁₅ ⁺;calc. 694.3141).

4′,6′-O-2-Thiophenylideneparomomycin (5.22). Reaction of 42 mg of 4.22gave 29 mg of 5.22 (82%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.37. IR (ATR): 3353w, 3287w, 3174w (br.), 2909w, 1595w, 1543w, 1445w,1377w, 1332w, 1243w, 1100s, 1053s, 1023s, 974s, 926s. HR-Maldi-MS:733.2763 (33, [M+Na+H]⁺, C₂₈H₄₈N₅NaO₁₄S⁺; calc. 733.2816); 732.2730(100, [M+Na]⁺, C₂₈H₄₇N₅NaO₁₄S⁺; calc. 732.2738).

4′,6′-O-Ethylideneparomomycin (5.23). Reaction of 18 mg of 4.23 gave 15mg of 5.23 (99%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1) 0.27. IR(ATR): 3286w (br.), 2888w, 1664w, 1571w, 1472w, 1392w, 1341w, 1102s,1017s, 995s, 937w, 907w. HR-Maldi-MS: 665.3048 (31, [M+Na+H]⁺,C₂₅H₄₈N₅NaO₁₄ ⁺; calc. 665.3095); 664.3018 (100, [M+Na]⁺, C₂₅H₄₇N₅NaO₁₄⁺; calc. 664.3012).

4,6′-O-(2-Phenyl-ethylideneparomomycin (5.24). Reaction of 52 mg of 4.24gave 39 mg of 5.24 (89%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.33. HR-Maldi-MS: 741.3349 (37, [M+Na+H]⁺, C₃₁H₅₂N₅NaO₁₄ ⁺; calc.741.3408); 740.3312 (100, [M+Na]⁺, C₃₁H₅₁N₅NaO₁₄ ⁺; calc. 740.3330).

4′,6′-O-(3-Phenyl)-propylideneparomomycin (5.25). Reaction of 52 mg of4.25 gave 35 mg of 5.25 (79%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.27. IR (ATR): 3356w (br.), 2870w, 1740w, 1594w, 1495w, 1454w, 1386w,1119s, 1016s, 931s. ¹H-NMR (500 MHz, D₂O, assignment based on a HSQCspectrum): 7.40-7.37 (m, 2 arom. H); 7.33-7.27 (m, 3 arom. H); 5.47 (d,J=3.8, H—C(1′)); 5.37 (d, J=2.5, H—C(1″)); 5.00 (d, J=1.8, H—C(1′″));4.68 (t, J=5.3, PhCH₂CH₂CH); 4.46 (dd, J=6.6, 5.0, H—C(3″)); 4.31 (dd,J=4.9, 2.5, H—C(2″)); 4.18-4.13 (m, 2H, H_(a)—C(6′), H—C(4″)); 4.07-4.04(m, 2H, H—C(5′″), H—C(3′″)); 3.90-3.85 (m, 2H, H_(a)—C(5″), H—C(5′));3.78-3.65 (m, 4H, H—C(3′), H—C(5), H_(b)—C(5″), H—C(4′″)); 3.62 (t,J=10.5, H_(b)—C(6′)); 3.47 (t, J=9.3, H—C(4)); 3.42 (t, J=9.6, H—C(4′));3.40 (t, J=9.7, H—C(6); 3.17 (dd, J=13.5, 8.5, H_(a)—C(6′″); 3.09-3.06(m, 2H, H_(b)—C(6′″), H—C(2′″)); 2.93 (ddd, J=12.2, 9.5, 4.1, H—C(3));2.86 (dd, J=10.0, 3.8, H—C(2′)); 2.82-2.75 (m, 2H, H—C(1), PhCH₂CH₂);2.03-1.95 (m, 3H, H_(eq)—C(2), PhCH₂CH₂); 1.25 (q, J=12.4, H_(ax)—C(2)).HR-Maldi-MS: 755.3505 (38, [M+Na+H]⁺, C₃₂H₅₄N₅NaO₁₄ ⁺; calc. 755.3565);754.3467 (100, [M+Na]⁺, C₃₂H₅₃N₅NaO₁₄ ⁺; calc. 754.3481); 732.3642 (38,[M+H]⁺, C₃₂H₅₄N₅O₁₄ ⁺; calc. 732.3667).

4′,6′-O-(3-Phenyl)-propenylideneparomomycin (5.26). Reaction of 24 mg of4.26 gave 19 mg of 5.26 (92%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.34. IR (ATR): 3353w, 3288w, 2915w, 1593w, 1493w, 1451w, 1377w, 1336w,1135s, 1115s, 1054s, 1022s, 994s, 970s. HR-Maldi-MS: 731.3519 (37,[M+2H]⁺, C₃₂H₅₃N₅O₁₄ ⁺; calc. 731.3589); 730.3492 (100, [M+H]⁺,C₃₂H₅₂N₅O₁₄ ⁺; calc. 730.3511).

4′,6′-O-Cyclohexylmethylideneparomomycin (5.27). Reaction of 26 mg of4.27 gave 16 mg of 5.27 (73%). White solid. R_(f) (MeOH/25% aq. NH₃ 4:1)0.25. IR (ATR): 3127m, 3029m, 2847w, 1753w, 1712w, 1613w, 1514w, 1441w,1399m, 1101s, 996s. HR-Maldi-MS: 711.3859 (34, [M+2H]⁺, C₃₀H₅₇N₅O₁₄ ⁺;calc. 711.3902); 710.3822 (100, [M+H]⁺, C₃₀H₅₆N₅O₁₄ ⁺; calc. 710.3824).

Synthesis of 4′- and 6′-O-ether Derivatives

6,3′,2″,5″,3′″,4′″-Hexa-O-acetyl-1,3,2′,2′″,6′″-pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′-O-benzylparomomycin(9). Under N₂, a soln. of 6 (1.8 g, 1.66 mmol) in dry CH₂Cl₂ (25 ml,dried over CaH₂) was cooled to −5° C., treated with 2M BH₃.Me₂S in THF(8.3 ml, 16.6 mmol) and 1M Bu₂BOTf in CH₂Cl₂ (0.83 ml, 0.83 mmol), andstirred for 5 h. After dilution with sat. NaHCO₃ soln. the layers wereseparated. The org. layer was washed with brine, dried (MgSO₄),filtered, and evaporated. FC (AcOEt/cyclohexane 9:11) gave 9 (1.1 g,61%). R_(f) (AcOEt/cyclohexane 1:1) 0.41. M.p. 82-86° C. [α]_(D)²⁵=+98.2 (c=0.13, MeOH). IR (ATR): 2940w, 2872w, 2100s, 1740s, 1492w,1453w, 1430w, 1371m, 1215s, 1027s. HR-ESI-MS: 1110.3489 (100, [M+Na]⁺,C₄₂H₅₃N₁₅NaO₂₀ ⁺; calc. 1110.3469). Anal. calc. for C₄₂H₅₃N₁₅O₂₀(1087.97): C, 46.37; H, 4.91; N, 19.31, O 29.41; found: C, 46.27; H,4.82; N, 19.02, O 29.38.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′-O-benzylparomomycin(10). Under N₂, a soln. of 9 (100 mg, 0.09 mmol) in 0.02N MeONa in MeOH(2 ml) was stirred at 26° C. for 12 h, and neutralized with Amberlite-IR120 (H⁺ form). Filtration, evaporation and FC (CHCl₃/AcOEt/MeOH10:17.5:2) gave 10 (65 mg, 86%). White solid. R_(f) (CHCl₃/AcOEt/MeOH4:9:1) 0.45. M.p. 98° C. [α]_(D) ²⁵=+106.1 (c=0.14, MeOH). IR (KBr):3418s, 2928m, 2107s, 1633m, 1497w, 1454m, 1384m, 1332m, 1261s, 1114m,1068m, 1029s, 939w, 749w. HR-ESI-MS: 858.2835 (100, [M+Na]⁺,C₃₀H₄₁N₁₅NaO₁₄ ⁺; calc. 858.2855). Anal. calc. for C₃₀H₄₁N₁₅O₁₄.0.3AcOEt(862.17): C, 43.46; H, 5.07; N, 24.37; found: C, 43.13; H, 5.03; N,24.47.

4′-O-Benzylparomomycin (11). A soln. of 10 (30 mg, 0.04 mmol) in THF (3ml) was treated with 0.1M aq. NaOH (1 ml) and 1M PMe₃ in THF. (0.22 ml,0.22 mmol) and stirred at 50° C. for 2 h. Evaporation and FC (MeOH/25%aq. NH₃ 4:3) gave 11 (20 mg, 79%). White solid. R_(f) (CHCl₃/MeOH/25%aq. NH₃ 1:3:4) 0.55. [α]_(D) ²⁵=+36.3 (c=0.11, H₂O). IR (KBr): 3418s,2925m, 2852w, 1631m, 1537w, 1452w, 1397w, 1384m, 1298w, 1123s, 1051s,947w. HR-ESI-MS: 728.3269 (100, [M+Na]⁺, C₃₀H₅₁N₅NaO₁₄ ⁺; calc.728.3330); 706.3493 (100, [M+H]⁺, C₃₀H₅₂N₅O₁₄ ⁺; calc. 706.3511).

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′,6′-O-benzylidene-6,3′,2″,5″,3′″,4′″-hexakis-O-(4-methoxybenzyl)paromomycin(12). Under Ar, a soln. of 4.1 (810 mg, 0.97 mmol) in THF (20 ml) wastreated with NaH (637 mg, 50-60% suspension in oil, ca 13.3 mmol),p-MeOBnCl (0.90 ml, 6.66 mmol), and Bu₄NI (110 mg), stirred at 0° C. for4 h and at 26° C. for 20 h, cooled to 0° C., and diluted portionwisewith H₂O. After evaporation, the aq. layer was extracted with AcOEt(3×20 ml). The combined org. layers were washed with brine, dried(MgSO₄), filtered, and evaporated. FC (AcOEt/cyclohexane 1:4) gave 12(722 mg, 48%). White solid. R_(f) (AcOEt/cyclohexane 1:1) 0.46. M.p.54.6-56.4° C. (softens at 46° C.). [α]_(D) ²⁵=+75.8 (c=0.40, CHCl₃). IR(CHCl₃): 3021w, 3006w, 2937w, 2839w, 2867w, 2106s, 1612m, 1586w, 1514s,1465w, 1442w, 1368w, 1302w, 1250s, 1174m, 1091m, 1035m, 847w, 823w.HR-ESI-MS: (100, [M+Na]⁺, C₇₈H₈₇N₁₅NaO₂₀ ⁺; calc. 1576.6149). Anal.calc. for C₇₈H₈₇N₁₅O₂₀ (1554.61): C, 60.26; H, 5.64; N, 13.51; found: C,60.23, H, 5.81, N, 13.31.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-6′-O-benzyl-6,3′,2″,5″,3′″,4′″-hexakis-O-(4-methoxybenzyl)paromomycin(13). Under N₂, a soln. of 12 (677 mg, 0.44 mmol) in THF (15 ml) wastreated with 4 Å molecular sieves, stirred for 1 h, cooled to 0° C.,treated with NaCNBH₃ (443 mg, 7.05 mmol) at 0° C., and then dropwisewithin 1 h with 0.7M HCl in Et₂O (14 ml, 9.80 mmol) (methyl orange wasadded to indicate the pH of solution) and stirred for 4 h. Afterneutralization with sat. NaHCO₃ soln. and evaporation, the aq. layer wasextracted with AcOEt (3×25 ml). The combined org. layers were washedwith brine, dried (MgSO₄), filtered, and evaporated. FC(AcOEt/cyclohexane 3:7) gave 13 (219 mg, 33%). White solid. R_(f)(AcOEt/cyclohexane 1:1) 0.42. M.p. 52-55° C. [α]_(D) ²⁵=+66.9 (c=0.37,CHCl₃). IR (CHCl₃): 3673w, 3020s, 2937w, 2839w, 2106s, 1612m, 1583w,1514m, 1465w, 1438w, 1366w, 1302w, 1250m, 1174w, 1118w, 1072w, 1035m,901w. HR-ESI-MS: 1578.6274 (100, [M+Na]⁺, C₇₈H₈₉N₁₅NaO₂₀ ⁺; calc.1578.6306); 1529.6556 (84, [M−N₂+2H]⁺, C₇₈H₉₁N₁₃NaO₂₀ ⁺; calc.1529.6503); 1528.6536 (94, [M−N₂+H]⁺, C₇₈H₉₀N₁₃NaO₂₀ ⁺; calc.1528.6425). Anal. calc. for C₇₈H₈₉N₁₅O₂₀ (1556.65): C, 60.18; H, 5.76;N, 13.50; found: C, 60.05; H, 5.64; N, 13.47.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-6′-O-benzylparomomycin(14). Under N₂, a soln. of 13 (107 mg, 0.07 mmol) in CH₂Cl₂/H₂O/i-PrOH20:1:1 (7 ml) was treated with DDQ (103 mg, 0.45 mmol) at 0° C. andstirred for 2 h and at 26° C. for 24 h. The colour of the mixturechanged from colourless to green and then to light orange. Afterneutralization with sat. NaHCO₃ soln., the aq. layer was extracted withAcOEt (3×20 ml). The combined org. layers were washed with brine, dried(MgSO₄), filtered, and evaporated. FC (CHCl₃/AcOEt/MeOH 10:17.5:2)) gave14 (34 mg, 59%). White solid. R_(f) (CHCl₃/AcOEt/MeOH 4:9:1) 0.68. M.p.89.1-92.9° C. [α]_(D) ²⁵=+97.2 (c=0.12, MeOH). IR (KBr): 3434s, 2929w,2107s, 1635w, 1500w, 1401w, 1384m, 1331w, 1262m, 1140m, 1077m, 1036m,743w. HR-ESI-MS: 858.2836 (100, [M+Na]⁺, C₃₀H₄₁N₁₅NaO₁₄ ⁺; calc.858.2855). Anal. calc. for C₃₀H₄₁N₁₅O₁₄.CH₃OH (867.78): C, 42.91; H,5.23; N, 24.21; found: C, 42.73; H, 4.96; N, 24.25.

6′-O-Benzylparomomycin (15). A soln. of 14 (25 mg, 0.03 mmol) in THF (3ml) was treated with 0.1M aq. NaOH (1 ml) and 1M PMe₃ in THF (0.22 ml,0.22 mmol) and heated to 50° C. for 2 h. Evaporation and FC (MeOH/25%aq. NH₃ 4:3) gave 15 (18 mg, 85%). White solid. R_(f) (CHCl₃/MeOH/25%aq. NH₃ 1:4:3) 0.56. M.p. 165° C. (dec.). [α]_(D) ²⁵=+28.1 (c=0.08,H₂O). IR (KBr): 3420s, 2925m, 1633m, 1595m, 1491w, 1454w, 1397w, 1384m,1252w, 1151m, 1092m, 1026s, 1051s, 741w. HR-ESI-MS: 728.3269 (100,[M+Na]⁺, C₃₀H₅₁N₅NaO₁₄ ⁺; calc. 728.3330); 706.3493 (100, [M+H]⁺,C₃₀H₅₂N₅O₁₄ ⁺; calc. 706.3511). Anal. calc. for C₃₀H₅₁N₅O₁₄.6 AcOH.3H₂O(1120.11)^(±): C, 45.04; H, 7.29; N, 6.25; found: C, 44.85; H, 6.72; N,6.68.

Typical procedure for the formation of ethers 16,17a-d (Scheme 5). (GP1): Under N₂, a soln. of alcohol (1 equ.) in DMF (c=0.1) was treatedwith NaH (1.5 equ. per hydroxyl group, 50-60% suspension in oil),followed by alkyl chloride (or bromide) (1.5 equ. per hydroxyl group)and Bu₄NI (0.1 equ.), stirred at 25° C. for 4-24 h, and diluted with H₂Oand Et₂O. After separation of phases, the aq. layer was extracted withEt₂O (3×). The combined org. layers were dried (MgSO₄) and evaporated.FC.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-6,3′,2″,5″,3′″,4′″-hexa-O-p-methoxybenzyl-4′-hydroxy-6′-O-monomethoxytritylparomomycin(16). Under N₂, a soln. of 12 (400 mg, 0.256 mmol) in MeOH/CH₂Cl₂ (10:1,5.5 ml) was treated with TsOH.H₂O (49 mg, 0.3 mmol), stirred at 25° C.for 3 h, and diluted with 1N NaOH. After separation of phases, the aq.layer was extracted with CH₂Cl₂ (3×5 ml). The combined org. layers weredried (MgSO₄) and evaporated. FC gave as a white solid1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-6′,4′-dihydroxy-6,3′,2″,5″,3′″,4′″-hexa-O-p-methoxybenzylparomomycin (296 mg, 79%), R_(f) (AcOEt/cyclohexane 1:1) 0.37, which wasreacted according to GP 1. Reaction gave 16 (1.319 mg, 65%). R_(f)(AcOEt/cyclohexane 3:7) 0.58. White solid, m.p. 60-65° C. [α]_(D)²⁵=+96.4 (c=0.12, CHCl₃). IR (ATR): 2927w, 2103s, 1611m, 1581w, 1513s,1460w, 1362w, 1303w, 1249s, 1173m, 1033s. HR-Maldi-MS: 1776.6813 (92,[M+K]⁺, C₉₁H₉₉N₁₅KO₂₁ ⁺; calc. 1776.6777); 1760.7034 (82, [M+Na]⁺,C₉₁H₉₉N₁₅NaO₂₁ ⁺; calc. 1760.7038). Anal. calc. for C₉₁H₉₉N₁₅O₂₁(1738.85): C, 62.86; H, 5.74; N, 12.08; found: C, 63.25; H, 6.06; N,11.55.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-6,3′,2″,5″,3′″,4′″-hexa-O-p-methoxybenzyl-4′-O-p-chlorobenzyl-6′-O-monomethoxytrityllparomomycin(17a). (GP 1) Reaction of 16 gave 17a (518 mg, 96%). R_(f)(AcOEt/cyclohexane 3:7) 0.54. White solid, m.p. 66-71° C. [α]_(D)²⁵=+94.1 (c=0.08, ChCl3). IR (atr): 2932w, 2099s, 1611m, 1585w, 1512s,1462w, 1359w, 1301w, 1245s, 1173m, 1110m, 1068m, 1031s. HRMaldi-MS:1900.6892 (57, [M+K]⁺, C₉₈H₁₀₄ClN₁₅KO₂₁ ⁺; calc. 1900.6857); 1884.7151(84, [M+Na]⁺, C₉₈H₁₀₄N₁₅ClNaO₂₁ ⁺; calc. 1884.7117). Anal. calc. forC₁₀₀H₁₀₉N₁₅O₂₁.1.5H₂O (1890.44): C, 62.26; H, 5.71; N, 11.11; found: C,62.39, H, 5.83, N, 10.73.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-6,3′,2″,5″,3′″,4′″-hexa-O-p-methoxybenzyl-4′-O-p-(trifluoromethyl)benzyl-6′-O-monomethoxytritylparomomycin(17b). (GP 1) Reaction of 16 gave 17b (441 mg, 85%). R_(f)(AcOEt/cyclohexane 3:7×2) 0.54. White solid, m.p. 60-76° C. [α]_(D)²⁵=+70.3) (c=0.26, ChCl3). Ir (ATR): 2934w, 2100s, 1611m, 1586w, 1512s,1463w, 1362w, 1325m, 1302m, 1245s, 1173m, 1111m, 1065s, 1030s.HR-Maldi-MS: 1934.7083 (84, [M+K]⁺, C₉₉H₁₀₄F₃N₁₅KO₂₁ ⁺; calc.1934.7120); 1918.7333 (80, [M+Na]⁺, C₉₉H₁₀₄N₁₅F₃NaO₂₁ ⁺; calc.1918.7381). Anal. calc. for C₉₉H₁₀₄N₁₅O₂₁F₃ (1896.97): C, 62.68, H,5.53, N, 11.08; found: C, 62.42; H, 5.62; N, 10.66.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-6,3′,2″,5″,3′″,4′″-hexa-O-p-methoxybenzyl-4′-O-propylphenyl-6′-O-monomethoxytritylparomomycin(17c). (GP 1) Reaction of 16 gave 17c (459 mg, 86%). R_(f)(AcOEt/cyclohexane 3:7) 0.6. White solid, m.p. 63-78° C. [α]_(D)²⁵=+56.9 (c=0.65, CHCl₃). IR (ATR): 2933w, 2099s, 1672w, 1611m, 1585w,1512s, 1454w, 1363w, 1301w, 1245s, 1174m, 1029s. HR-Maldi-MS: 1894.7513(72, [M+K]⁺, C₁₀₀H₁₀₉N₁₅KO₂₁ ⁺; calc. 1894.7560); 1878.7773 (82,[M+Na]⁺, C₉₁H₉₉N₁₅NaO₂₁ ⁺; calc. 1878.7820). Anal. calc. forC₁₀₀H₁₀₉N₁₅O₂₁ (1857.02): C, 64.68; H, 5.92; N, 11.31; found: C, 65.08;H, 5.71; N, 10.86.

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-6,3′,2″,5″,3′″,4′″-hexa-O-p-methoxybenzyl-4′-O-benzyloxymethyl-6′-O-monomethoxytrityliparomomycin(17d). (GP 1) Reaction of 16 gave 17d (442 mg, 83%). R_(f)(AcOEt/cyclohexane 3:7) 0.54. White solid, m.p. 52-55° C. IR (ATR):2933w, 2001s, 1611m, 1585w, 1513s, 1463w, 1361w, 1301m, 1247s, 1174m,1111m, 1068m, 1033s. HR-Maldi-MS: 1896.7326 (16, [M+Na]⁺, C₉₉H₁₀₇N₁₅KO₂₂⁺; calc. 1896.7352); 1880.7566 (22, [M+Na]⁺, C₉₉H₁₀₇N₁₅NaO₂₂ ⁺; calc.1880.7613); 1866.7181 (65, [M−CH₃O+K]⁺, C₉₈H₁₀₅N₁₅KO₂₁ ⁺; calc.1866.7247); 1850.7403 (89, [M—CH₃O+Na]⁺, C₉₈H₁₀₅N₁₅NaO₂₁ ⁺; calc.1850.7507).

General procedure for deprotection of protected derivatives to givecompounds 19a-d: (GP 2): Under N₂, a soln. of protected derivative 17 (1equiv.) in CH₂Cl₂/MeOH/H₂O 20:1:0.4 (c=0.02) was treated with DDQ (1.1equiv. per group), stirred at 25° C. for 12-24 h, and neutralized with1N NaOH, diluted with AcOEt. After separation of phases, the aq. layerwas extracted with AcOEt (3×). The combined org. layers were dried(MgSO₄) and evaporated. FC. A soln. of azido compound (1 equiv.) inTHF/0.1M NaOH 4:1 (c=0.03) was treated with 1M PMe₃ in THF (1.2 equiv.per azido group) and stirred for 4-8 h at 50° C. After evaporation, FC.

4′-p-chlorobenzylparomomycin (19a). (GP 2) Reaction of 17a gave azidointermediate 18a (120 mg, 54%) as a white solid. R_(f) (MeOH/CHCl₃/AcOEt0.5:2:2) 0.29. Final compound 19a (47 mg, 86%). R_(f) (MeOH/NH_(3aq.)8:2) 0.27. White solid, m.p. 145° C. (decomp.). [α]_(D) ²⁷=+60.3(c=0.27, H₂O). IR (atr): 3358m, 2915m, 1590w, 1454w, 1366w, 1088s,1014s. HR-Maldi-MS: 762.2921 (100, [M+Na]⁺, C₃₀H₅₀ClN₅NaO₁₄ ⁺; calc.762.2940); 740.3110 (86, [M+H]⁺, C₃₀H₅₁ClN₅O₁₄ ⁺; calc. 740.3121).

4′-p-(trifluoromethyl)benzylparomomycin (19b). (GP 2) Reaction of 17bgave azido intermediate 18b (130 mg, 80%) as a white solid. R_(f)(MeOH/CHCl₃/AcOEt 0.5:2:2) 0.26. Final compound 19b (84 mg, 87%). R_(f)(MeOH/NH_(3aq.) 8:2) 0.30. White solid, m.p. 148° C. (dec.). [α]_(D)²⁵=+65.4 (c=0.16, H₂O). IR (ATR): 3156m, 2921m, 1551m, 1403m, 1326s,1107s, 1064s, 1016s. HR-Maldi-MS: 796.3201 (72, [M+Na]⁺, C₃₁H₅₀F₃N₅NaO₁₄⁺; calc. 796.3204); 774.3366 (100, [M+H]⁺, C₃₁H₅₁F₃N₅O₁₄ ⁺; calc.774.3385).

4′-p-(3-phenylpropyl)paromomycin (19c). (GP 2) Reaction of 17c gaveazido intermediate 18c (105 mg, 74%) as a white solid. R_(f)(MeOH/CHCl₃/AcOEt 0.5:2:2) 0.40. Final compound 19c (64 mg, 92%). R_(f)(CHCl₃/MeOH/NH_(3aq.) 1:3:2) 0.47. White solid, m.p. 179° C. (dec.).[α]_(D) ²⁵=+63.0 (c=0.12, H₂O). IR (ATR): 3191w, 2920w, 1572w, 1495w,1454w, 1381w, 1339w, 1015s. HR-Maldi-MS: 756.3648 (37, [M+Na]⁺,C₃₂H₅₅N₅NaO₁₄ ⁺; calc. 756.3643); 734.3805 (100, [M+H]⁺, C₃₂H₅₆N₅O₁₄ ⁺;calc. 734.3824).

4′-Benzyloxymethylparomomycin (19d). (GP 2) Reaction of 17d gave azidointermediate 18d (70 mg, 72%) as a white solid. R_(f) (MeOH/CHCl₃/AcOEt0.5:2:2) 0.31. Final compound 19d (36 mg, 72%). R_(f)(CHCl₃/MeOH/NH_(3aq.) 1:3:2) 0.36. White solid, m.p. 143° C. (decomp.).[α]_(D) ²⁷=+48.9 (c=0.24, H₂O). IR (ATR): 3031m, 1622w, 1525w, 1429m,1043s. HR-Maldi-MS: 758.3428 (20, [M+Na]⁺, C₃₁H₅₃N₅NaO₁₅ ⁺; calc.758.3436); 736.3598 (100, [M+H]⁺, C₃₁H₅₄N₅O₁₅ ⁺; calc. 736.3616).

1,3,2′,2′″,6′″-Pentadeamino-1,3,2′,2′″,6′″-pentaazido-4′-O-p-methoxybenzylparomomycin(18e). Under N₂, 4.2 (204 mg, 0.236 mmol) cooled to −5° C. was treatedwith 1M BH₃.THF (2.36 ml) and 1M Bu₂BOTf in CH₂Cl₂ (0.236 ml), andstirred at −5° C.-0° C. for 50 min. After completion Et₃N (0.1 ml) andMeOH (0.1 ml) were added. Reaction mixture was coevaporated with MeOHthree times. FC gave 18e (120 mg, 59%). R_(f) (AcOEt/CHCl₃/MeOH2:2:0.25) 0.55. White solid, m.p. 65-68° C. [α]_(D) ²⁵=+99.3 (c=1.63,MeOH). IR (ATR): 3374m, 2942w, 2874w, 2102s, 1770w, 1631w, 1612w, 1514w,1453w, 1369w, 1331w, 1248m, 1143w, 1101m, 1075m, 1027s. HR-Maldi-MS:888.2942 (100, [M+Na]⁺, C₃₁H₄₃N1₅NaO₁₅ ⁺; calc. 888.2961); 456.0339 (91,[M+H]²⁺+Na, C_(15.5)H₂₂N_(7.5)NaO_(7.5) ²⁺; calc. 456.1468).

4′-p-Methoxybenzylparomomycin (19e). A soln. of 18e (60 mg, 0.058 mmol)in THF (2 ml) was treated with 0.1M NaOH soln. (0.5 ml) and 1M PMe₃ inTHF (0.346 ml) and stirred for 4 h at 50° C. After evaporation, FC andcoevaporation with 10% AcOH gave 19e as an acetic salt (44 mg, 74%)R_(f) (MeOH/CHCl₃/NH_(3aq.) 1:3:2) 0.33. White solid, m.p. 190° C.(dec.). [α]_(D) ²⁵=+48.93 (c=0.32, H₂O). IR (ATR): 3115m(br), 2874w,1609w, 1513m, 1405m, 1333w, 1248w, 1050s, 1031s. HR-Maldi-MS: 736.3597(100, [M+H]⁺, C₃₁H₅₄N₅O₁₅ ⁺; calc. 736.3616).

Example: 2 Biological Assay

In the following the biological assay used for determining the activityof the compounds of the invention as exemplified in example 1 above toselectively target bacterial 16S ribosomal RNA and not to target at allor target to a substantially less degree eukaryotic cytosolic and/ormitochiondrial ribosomes is described in detail.

Production of Recombinant M. smegmatis Strains

The strain used for introduction/selection of mutational alterations wasa genetically modified derivative of Mycobacterium smegmatis carrying asingle functional chromosomal rRNA operon (Sander et al. Mol. Microbiol.1996, 22: 841-848). This strain, termed M. smegmatis rrn⁻, allowed forthe selection of mutational rRNA gene alterations using a plasmidcarrying the rRNA gene with the respective mutational alteration.)

The rRNA gene carried on the plasmid encodes either the complete rRNAoperon (Prammananan et al. Antimicrob. Agents Chemother. 1999, 43:447-453) or a non-functional rRNA gene fragment of approximately 1.0 kb(Pfister et al. Antimicrob. Agents Chemother. 2003, 47: 1496-1502).PCR-mutagenesis in vitro was used to generate the mutagenized rRNA genefragment. In the case of a partial rRNA gene fragment the mutagenizedrRNA gene fragment was cloned into vectors pMV261 or pMV361 (Sander etal. Mol. Microbiol. 2002, 46: 1295-1304; Pfister et al. Antimicrob.Agents Chemother. 2003, 47: 1496-1502) to result in vectors carrying apartial rRNA gene fragment of approximately 1.0 kb with the mutationalalteration introduced. In the case of plasmids carrying the completerRNA operon the mutagenized rRNA gene fragment obtained by PCR wascloned into vectors pMV361 rRNA or pMV261 rRNA using appropriaterestriction sites (Prammananan et al. Antimicrob. Agents Chemother.1999, 43: 447-453). Vectors pMV361 rRNA and pMV261 rRNA carried acomplete copy of the rRNA operon from M. smegmatis (Sander et al. Mol.Microbiol. 1997, 26: 469-480). Introduction of mutations into theplasmid encoded rRNA was confirmed by sequencing.

The single rRNA allelic derivative of M. smegmatis, i.e. M. smegmatisrrn, was used for transformation of the plasmids. The strain was madeelectro-competent and transformed according to standard techniques andas described previously (Sander et al. Mol. Microbiol. 1997, 26:469-484). Following primary selection, the plasmid-encoded mutationalrRNA gene alteration was transferred into the single chromosomal rRNAoperon by means of RecA-mediated homologous recombination (Prammanananat al. Antimicrob. Agents Chemother. 1999, 43: 447-453). Introduction ofthe mutational rRNA gene alteration into the single functionalchromosomal rRNA operon by gene conversion was confirmed by sequencedetermination.

In another aspect of the technique, the single functional chromosomalrRNA operon was inactivated and the synthesis of ribosomal RNA wasdriven exclusively by the mutated plasmid-encoded rRNA operon.

The following demonstrates one way of producing a strain with allendogenous rrn genes deleted and with functional ribosomal RNA producedby a plasmid encoded rRNA operon.

A combination of positive, e.g. aph, and negative-selectable markers,e.g. sacB, was used for unmarked deletion mutagenesis. In brief, thesacB gene was cloned into the mycobacterial expression vector pMV361(Stover et al. Nature 1991, 351:456-460) to result in pMS32a. In pMS32a,sacB is located downstream of the hsp60 promoter. A restriction fragmentof pMS32a carrying the hsp60p-sacB construct was transferred into thecloning vector pGEM-7 (Promega) to result in plasmid pZ130 which wasused as backbone in the construction of both rrnA and rrnB replacementvectors. Chromosomal DNA sequences flanking the 5′ and 3′ region of eachrrn operon were obtained by PCR and cloned into pZ130. Following furthermodifications, the rm replacement vectors were obtained. As example, thegeneration of the rrnB replacement vector is described here and thestrategy for inactivation of rrnB illustrated in FIG. 1.

For complementation, a functional rmB operon was cloned into anintegration-proficient vector. Following the strategy outlined in FIG. 2a derivative of M. smegmatis, i.e. strain ΔrrnA ΔrrnB attB::prrnB, wasobtained where both endogenous chromosomal rRNA operons are inactivatedby gene deletion across the entire 16S, 23S, and 5S rRNA genes (rrs,rrl, and rrf, see FIG. 1). This strain is completely devoid ofchromosomal rRNA genes and rRNA is exclusively transcribed from plasmidDNA.

Such strains are particularly useful because they avoid the interferenceof the bacterial cell's own naturally occurring ribosomal activity withthat of the introduced at least partially heterologous, mitochondrial orcytosolic bacterial ribosomes in the assay methods of the presentinvention.

Ribosomal drug susceptibility was studied by determining the minimalinhibitory concentrations, as described in detail in Pfister et al. 2003and 2005 (see above).

The recombinants carrying the respective mutational alterations in thefunctional rRNA operon were colony-purified and subjected todeterminations of minimal inhibitory concentrations (MIC) to determineribosomal drug susceptibility. Cultures from single colonies were grownin LB medium supplemented with 0.05% Tween 80 and used for MIC tests ina microtiter plate format. Starting cultures contained 200 μl ofbacterial cells at an optical density of 0.025 at 600 nm, and therespective drug was added in twofold series of dilution. The MIC wasdefined as the drug concentration at which the growth of the cultureswas completely inhibited after 72 h of incubation at 37° C.,corresponding to 24 generations.

Key nucleotides which distinguish the prokaryotic from the eukaryoticdecoding site are 16S rRNA positions 1408 (bacterial ribosome: A,eukaryotic cytosolic ribosomes: G) and 1491 (bacterial ribosomes: G,eukaryotic cytosolic ribosomes: A, eukaryotic mitochondrial ribosome:C).

As demonstrated by the results in table 1 below the compounds of theinvention are highly selective for bacterial ribosomal RNA. Hence, theyare highly effective in antibiotic therapy of bacterial infection inmammals, in particular humans and they can be dosed higher than currentnon-selective antibiotics that have to be dosed much lower in order toavoid damage to the mammal's mitochondrial and cytosolic RNA.

TABLE 1 Activity of selected compounds and comparators towards bacterialand mutant ribosomes carrying key nucleotides of the deconding site asdetermined by investigating minimal inhibitory concentrations (mg/ml)ΔrrnB ΔrrnB 1408G ΔrrnB 1491A ΔrrnB 1491C μg/ml μg/ml μg/ml μg/mlParomomycin 1 64 64 ≧512 Neomycin B 0.5-1   >512 4 16-32 5.1  4 512512 >512 5.2  4 >512 >512 >512 5.3  4-8 256-512 512 >512 5.4  8-16 >512 >512 >512 5.5  4-8 ≧512 >512 >512 5.6  4 512 >512 >512 5.7 2-4 256-512 256 >512 5.8  4 >512 >512 >512 5.9  4 128-256 128-256 1285.10 16-32 >512 >512 >512 5.11 64 128  64-128 64 5.12 2-4 >512 >512 >5125.13 32 >512 >512 >512 5.14 32-64 >512 >512 >512 5.15 2-4 256-512 512512 5.16  4-16 512 512 >512 5.17 2-4 >512 512 512 5.18 2-4 256-512256 >512 5.19 4-8 512 256-512 256 5.20 2-4 >512 512 512 5.21 8-16 >512 >512 >512 5.22  8-16 >512 >512 >512 5.23 32 >512 >512 >5125.24 64 256 256 128-256 5.25 1 512 ≧512 ≧512 5.26 2-4 ≧512 ≧512 ≧5125.27 32-64 >512 >512 >512 5.28 4 512 512 >512 5.29 4-8 >512 >512 >51211   4 >512 ≧512 >512 15   32-64 >512 >512 >512 19a   2 ≧512 ≧512 >51219b   8 >512 >512 ≧512 19c    8-16 ≧512 ≧512 512 19d   16 >512 >512 >51219e   8 >512 >512 >512

Example 3 Antibiotic Resistance

Selected compounds of the present invention were tested for antibioticsusceptibility using clinical isolates of Staphylococci, Enterococci andEscherichia coli as well as strains carrying defined resistancedeterminants and compared to well known antibiotics of the state of theart. Drug susceptibility was determined in standard MIC assays, i.e. bydetermination of the minimal drug concentration required to inhibitbacterial growth in vitro.

The results indicate:

-   1. The selected compounds are active against clinical isolates with    resistance to standard aminoglycosides-   2. The selected compounds are not modified by defined aminoglycoside    resistance determinants, e.g. ANT4′, AAC6′, APH2″, AAC3, ANT2″

The results are summarized in table 2 below, where the numberscorrespond to MIC values in μg/ml.

TABLE 2 Minimal inhibitory concentrations (mg/l) of comparator compoundsagainst clinical bacterial isolates and strains with defined resistancedeterminants Gentamicin Kanamycin A Tobramycin Neomycin ParomomycinAG011 Staph. clinical 4 16 8 4 8 aureus isolate AG013 Staph. clinical≧256 ≧256 ≧256  64-128 ≧256 epi. isolate AG014 Staph. clinical ≧256 ≧256256 32 ≧256 epi. isolate AG016 S. aureus BM3002 1 4-8 0.5-1   1-2 2-4AG015 S. aureus BM3002 4 256 ≧256 128 >256 (ANT4′) AG017 E. faecalisJH2-2 8-16 32-64 16-32 64 128-256 AG019 E. faecalisJH2-2 >256 >256 >256 >256 >256 (APH2″ AAC6′) AG001 E. coli clinical 4 164 isolate AG002 E. coli clinical 2 16 4 isolate AG003 E. coli clinical≧256 32 32 isolate AG004 E. coli clinical >256 64 64 isolate AG006 E.coli BM13 4 8 2 AG007 E. coli BM13 128 8 8 (AAC3) AG008 E. coli BM13 64128 64 (ANT2″) AG009 E. coli BM13 32 >256 128-256 AAC(6′)- 1B 11 5.1 5.85.25 5.17 AG011 Staphylococcus clinical 8 8 4-8 4 4 aureus isolate AG013Staphylococcus clinical 16  8-16 8 4-8 4-8 epidermidis isolate AG014Staph. clinical 8 8  8-16 4 4 epi. isolate AG016 Staph. BM3002 16-3216-32 8 8 8 aureus AG015 Staph. BM3002 32 32-64 16  8-16 32 aureus(ANT4′) AG017 Enterococcus JH2-2 32-64 32-64 16-32 8 32 faecalis AG019E. faecalis JH2-2 >256 256 64 32 128 (APH2″ AAC6′) AG001 E. coliclinical 64 64 32 32-64 32-64 isolate AG002 E. coli clinical 32-64 64 —32-64 32-64 isolate AG003 E. coli clinical 32-64 32 — 32 32-64 isolateAG004 E. coli clinical 64 32-64 16-32 32 32 isolate AG006 E. coli BM1332 32 64 16-32 32 AG007 E. coli BM13 32 32 64 16-32 32 (AAC3) AG008 E.coli BM13  64-128 32 64 32 32 (ANT2″) AG009 E. coli BM13  64-128 128 64 64-128  64-128 AAC(6′)- 1B 5.26 5.20 19c 19a AG011 Staphylococcusclinical 4 1-2  — 2 aureus isolate AG013 Staphylococcus clinical 4-8 14-8 4 epidermidis. isolate AG014 Staph. clinical 4 1 2 2 epi isolateAG016 Staph. BM3002 8 4 4 4 aureus AG015 Staph. BM3002 16-32 8-16 168-16 aureus (ANT4′) AG017 Enterococcus JH2-2 16-32 8-16 8-16 8-16faecalis AG019 E. faecalis JH2-2  64-128 8-16 32 32-64  (APH2″ AAC6′)AG001 E. coli clinical 32-64 8 16-32  8-16 isolate AG002 E. coliclinical 64 — 16 16 isolate AG003 E. coli clinical 32 — 8-16 8-16isolate AG004 E. coli clinical 32 8 16 8-16 isolate AG006 E. coli BM1332 8-16 8-16 8-16 AG007 E. coli BM13 32 16-32  8-16 8-16 (AAC3) AG008 E.coli BM13 32 16 8-16 8-16 (ANT2″) AG009 E. coli BM13 128 16-32  32 32AAC(6′)- 1B

1-56. (canceled)
 57. Compounds of formula (I):

wherein: X, Y and Z denote in each case, independently of one another,—O—, —NH—, —S—, substituted or unsubstituted —CH₂— or a direct bond toR¹ and/or R²; R¹ and R² denote in each case, independently of oneanother, hydrogen, linear or branched, substituted or non-substitutedalkyl, alkenyl, alkynyl, alkylidene, carbocycle or YR¹ and ZR² togetherform a substituted or non-substituted cycloalkyl or a correspondingheterocyclic ring, with the proviso that R¹ and R² are not both H, R³and R⁴ denote in each case, independently of one another, hydrogen,amino or hydroxyl; R⁵ denotes glycosyl residues; and R⁶ denotes hydrogenand their diastereoisomers or enantiomers in the form of their bases orsalts of physiologically compatible acids.
 58. Compounds according toclaim 57, wherein R¹ and R² denote, in each case independently of oneanother, hydrogen, linear or branched, substituted or non-substitutedC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, alkynyl, or alkylidene, C₃-C₁₂ cycloalkyl,C₃-C₂₀ aryl, preferably aralkyl, C₃-C₂₀ heteroaryl or C₃-C₂₀heterocyclic.
 59. Compounds according to claim 57, wherein Y and Z areoxygen, preferably X, Y and Z are oxygen.
 60. Compounds according toclaim 57, wherein YR¹ is not NH₂ and/or ZR² is not OH, preferably YR¹and ZR² together form a substituted or non-substituted cycloalkyl or acorresponding heterocyclic ring, more preferably YR¹ and ZR² togetherform a 6-membered 4′,6′-cycloalkyl or substituted 4′,6″-cycloalkyl ring.61. Compounds according to claim 57, wherein R¹ comprises, preferablyis, a substituted or non-substituted (C₁-C₅ alkyl)aryl group and R² ishydrogen and/or R² is a linear or branched, substituted ornon-substituted C₁-C₇ alkyl, C₂-C₇ alkenyl, alkynyl, or alkylidene,C₃-C₇ cycloalkyl, C₃-C₇ aryl, preferably aralkyl, C₃-C₇ heteroaryl orC₃-C₇ heterocyclic and R¹ is hydrogen.
 62. Compounds according to claim57, wherein R³ is hydroxyl and/or R⁴ is amino.
 63. Compounds accordingto claim 57, wherein R⁵ is selected from the group consisting of mono-and polysaccharides, preferably a mono-, di- or trisaccharide, morepreferably a mono- or disaccharide, most preferably a disaccharide,especially preferred a2,6-diamino-2,6-dideoxy-β-L-idopyranosyl-(1→3)-β-D-ribofuranosyl moietyor a 3-amino-3-deoxy-α-D-glucopyranosyl moiety.
 64. Compounds accordingto claim 57 of formula (II):

wherein: X, Y and Z denote in each case, independently of one another,—O—, —NH—, —S—, substituted or non-substituted —CH₂—, preferably X, Yand Z are all oxygen; R⁷ denotes hydrogen, linear or branched,substituted or non-substituted alkyl, alkenyl, alkynyl, alkylidene, orcarbocycle; R⁸ denotes hydrogen, OH with the proviso that the compoundis stable, NH₂, NR_(a)R_(b), SH, SR_(a), OR_(a) or a linear or branched,substituted or non-substituted C₁-C₈ alkyl, preferably C₁-C₄ alkyl,wherein R_(a) and R_(b) are in each case, independently of one another,C₁-C₈ alkyl, preferably C₁-C₄ alkyl; R³ denotes hydrogen, amino orhydroxyl, preferably amino or hydroxyl; R⁴ denotes hydrogen, amino orhydroxyl, preferably amino or hydroxyl; R⁵ denotes a mono- orpolysaccharide, preferably a mono-, di- or trisaccharide, morepreferably a mono- or disaccharide, most preferably a disaccharide,especially preferred2,6-diamino-2,6-dideoxy-β-L-idopyranosyl-(1→3)-β-D-ribofuranosyl moiety;R⁶ denotes hydrogen.
 65. Compounds according to claim 64, wherein R⁷denotes hydrogen, a linear or branched, preferably linear, substitutedor non-substituted C₁-C₈ alkyl, preferably a substituted linear C₁-C₃alkyl, more preferably an aryl-substituted C₁-C₃ alkyl, most preferablyan aryl substituted ethyl group; a substituted or non-substituted C₃-C₈cycloalkyl, C₅-C₂₀ aryl, preferably C₅-C₁₂ aryl, C₅-C₂₀ heteroaryl,preferably C₅-C₁₂ heteroaryl, more preferably a substituted C₅-C₁₂heteroaryl, most preferably a (C₁-C₇ alkyl)aryl group; and wherein R⁷preferably comprises a halogen, preferably a chlorine, bromine, fluorineor iodine, more preferably a fluorine
 66. Compounds according to claim64, wherein R⁸ is selected from the group consisting of hydrogen,halogen or linear or branched, substituted or non-substituted C₁-C₈alkyl, preferably C₁-C₄ alkyl, most preferably hydrogen.
 67. Compoundsaccording to claim 57, wherein R¹ and R² denote a linear or branched,substituted or non-substituted alkyl or cycloalkyl, wherein one or moreof the carbon atoms are replaced, independently of one another, byoxygen, sulfur or nitrogen.
 68. Compounds according to claim 57 selectedfrom the group consisting of: 4′,6′-O-benzylideneparomomycin,4′,6′-O-p-methoxybenzylideneparomomycin,4′,6′-O-m-methoxybenzylideneparomomycin tetraacetate,4′,6′-O-o-methoxybenzylideneparomomycin,4′,6′-O-2,5-dimethoxybenzylideneparomomycin,4′,6′-O-p-nitrobenzylideneparomomycin,4′,6′-O-m-nitrobenzylideneparomomycin triacetate,4′,6′-O-p-chlorobenzylideneparomomycin,4′,6′-O-3,5-dichlorobenzylideneparomomycin,4′,6′-O-p-cyanobenzylideneparomomycin,4′,6′-O-p-phenylbenzylideneparomomycin,4′,6′-O-p-fluorobenzylideneparomomycin,4′,6′-O-3,5-dimethoxybenzylideneparomomycin,4′,6′-O-3,4,5-trimethoxybenzylideneparomomycin,4′,6′-O-m-chlorobenzylideneparomomycin,4′,6′-O-o-nitrobenzylideneparomomycin,4′,6′-O-p-trifluoromethylbenzylideneparomomycin,4′,6′-O-p-dimethylaminobenzylideneparomomycin,4′,6′-O-1-naphthylideneparomomycin, 4′,6′-O-2-naphthylideneparomomycin,4′,6′-O-2-furanylideneparomomycin, 4′,6′-O-2-thiophenylideneparomomycinand 4′,6′-O-ethylideneparomomycin,4′,6′-O-(2-phenyl)-ethylideneparomomycin,4′,6′-O-(3-phenyl)-propylideneparomomycin,4′,6′-O-(3-phenyl)-propenylideneparomomycin,4′,6′-O-cyclohexylmethylideneparomomycin, 4′-O-benzylparomomycin,6′-O-benzylparomomycin, 4′-p-chlorobenzyl paromomycin,4′-p-(trifluoromethyl)benzylparomomycin, 4′-benzyloxymethylparomomycinand 4′-p-methoxybenzylparomomycin.
 69. Use of one or more compoundsaccording to claim 57 for preparing a medicament, a medicament for thetreatment and/or prevention of a microbial infection, preferably abacterial infection, trypanosomiasis or leishmaniasis. 70.Pharmaceutical composition, comprising as active substance one or morecompounds according to claim 57 or pharmaceutically acceptablederivatives or prodrugs thereof, optionally combined with conventionalexcipients and/or carriers.
 71. Method for preparing a compoundaccording to claim 57, comprising one or more of the following steps: a)providing a compound according to the formula III:

wherein: V and W denote in each case, independently of one another, —O—,—NH— and —S—; R³ and R⁴ denote in each case, independently of oneanother, hydrogen, amino or hydroxyl; R⁵ and R⁶ denote in each case,independently of one another, hydrogen or glycosyl residues; b)protecting one or more, preferably all, of the amino groups; c)optionally protecting one or more, preferably all, of the hydroxygroups; d1) selectively transforming the optionally protected 4′- and/or6′-hydroxy groups to YR¹ and/or ZR²-groups of formula I or the ringsystem of formula II; or d2) selectively deprotecting the protected 4″-and/or 6″-hydroxy groups and selectively transforming the deprotected4′- and/or 6′-hydroxy groups to YR¹ and/or ZR²-groups of formula I orthe ring system of formula II; e) deprotecting the one or more aminogroups; f) deprotecting the one or more hydroxy groups; wherein theorder of steps b) and c) as well as e) and f) may be reversed, the orderof b) and c) as well as e) and f) being preferred.